Uses of mda-6

ABSTRACT

This invention provides a method of generating a subtracted cDNA library of a cell comprising: a) generating a cDNA library of the cell; b) isolating double-stranded DNAs from the cDNA library; c) releasing the double-stranded cDNA inserts from the double-stranded DNAs; d) denaturing the isolated double-stranded cDNA inserts; e) hybridizing the denatured double-stranded cDNA inserts with a labelled single-stranded nucleic acid molecules which are to be subtracted from the cDNA library; and f) separating the hybridized labeled single-stranded nucleic acid molecule from the double-stranded cDNA inserts, thereby generating a subtracted cDNA library of a cell. This invention also provides different uses of the subtracted library.

The invention described herein was supported in part by National CancerInstitute grants CA35675 and CA43208. The United States Government hascertain rights in this invention.

This application is a continuation-in-part of U.S. application Ser. No.08/143,576 filed Oct. 27, 1993 now U.S. Pat. No. 5,643,761, the contentsof which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced bywithin parentheses. Full citations for these publications may be foundat the end of each series of experiments. The disclosures of thesepublications in their entireties are hereby incorporated by referenceinto this application in order to more fully describe the state of theart as known to those skilled therein as of the date of the inventiondescribed and claimed therein.

Malignant melanoma is increasing at a rapid rate in North Americanpopulations and it is estimated that 1 in 100 children currently bornmay eventually develop superficial spreading-type melanoma (1). Althoughreadily curable at early stages, surgical and chemotherapeuticintervention are virtually ineffective in preventing metastatic diseaseand death in patients with advanced states of malignant melanoma (1).These observations emphasize the need for improved therapeuticapproaches to more efficaciously treat metastatic melanoma. Apotentially useful therapeutic modality for this and other malignanciescould involve the use of agents capable of inducing an irreversible lossin proliferative capacity in tumor cells without the requirement fordirect cytotoxicity, that is, the differentiation therapy of cancer(2-5). In previous studies, applicants have demonstrated that it ispossible to reprogram human melanoma cells to undergo terminal celldifferentiation with a concomitant loss of proliferative capacity bytreatment with the combination of recombinant human fibroblastinterferon (IFN-β) plus the antileukemic compound mezerein (MEZ) (6,7).The combination of IFN-β+MEZ induces terminal differentiation inmelanoma cells, innately resistant to the antiproliferative effect ofeither agent alone, and in human melanoma cells selected for resistanceto growth suppression induced by IFN-β (6,7). In contrast, treatmentwith IFN-β or MEZ alone results in the development of specificcomponents of the differentiation program in human melanoma cells, butthese agents do not induce most melanoma cells to undergo terminal celldifferentiation (6-8).

Terminal differentiation induced by IFN-β plus MEZ in human melanomacells is associated with an increase in melanin synthesis, changes incellular morphology (characterized by the production of dendrite-likeprocesses), modifications in cell surface antigenic profile, and anirreversible loss of proliferative capacity (3, 6-10). When usedseparately, IFN-β and MEZ induce both growth suppression and increasedmelanin synthesis and MEZ induces the production of dendrite-likeprocesses in specific human melanoma cells (6,8). Trans-retinoic acid(RA) is effective in inducing tyrosinase activity and enhancing melaninsynthesis in specific human melanoma cultures without altering cellgrowth, whereas mycophenolic acid (MPA) can induce growth suppression,increased tyrosinase activity and melanin synthesis, and dendriteformation (11). In contrast, the combination of IFN-β+recombinant immuneinterferon (IFN-γ results in a synergistic suppression in the growth ofhuman melanoma cells without inducing enhanced melanin synthesis ormorphologic changes (10,12). These observations suggest that the variouschanges induced during the process of differentiation in human melanomacells, that is, increased tyrosinase activity and melanin synthesis,antigenic changes, dendrite formation, and growth suppression, can occurwith and without the induction of terminal cell differentiation.

An unresolved issue is the nature of the gene expression changes thatoccur in human melanoma cells reversibly committed to differentiationvs. human melanoma cells irreversibly committed to terminaldifferentiation. This information will be important in defining on amolecular level the critical gene regulatory pathways involved in growthand differentiation in human melanoma cells. To begin to address thesequestions, applicants have used various experimental protocols thatresult in either growth suppression without the induction ofdifferentiation-associated properties, a reversible induction ofdifferentiation-associated traits, or terminal cell differentiation inthe HO-1 human melanoma cell line. As potential target genes relevant tothese processes, applicants have analyzed early growth response,extracellular matrix, extracellular matrix receptor, andinterferon-responsive genes. No unique gene expression change wasobserved solely in HO-1 cells induced to terminally differentiate vs.cultures reversibly growth arrested. However, treatment of HO-1 cellswith IFN-β+MEZ was associated with specific patterns of gene expressionchanges that were also apparent in HO-1 cells cultured in conditionedmedium obtained from terminal-differentiation-inducer-treated HO-1cells. Exposure to either the terminal differentiation- inducingcompounds or -conditioned medium resulted in the enhanced expression ofHLA Class I antigen, melanoma growth stimulatory activity (gro-MGSA),interferon-stimulated gene-15 (ISG-15), and fibronectin. Theseobservations support the potential involvement of a type I interferonand a gro/MGSA autocrine loop in the chemical induction ofdifferentiation in HO-1 cells

SUMMARY OF THE INVENTION

This invention provides a method of generating a subtracted cDNA libraryof a cell comprising: a) generating a cDNA library of the cell; b)isolating double-stranded DNAs from the cDNA library; c) releasing thedouble-stranded cDNA inserts from the double-stranded DNAs; d)denaturing the isolated double-stranded cDNA inserts; e) hybridizing thedenatured double-stranded cDNA inserts with a labelled single-strandednucleic acid molecules which are to be subtracted from the cDNA library;and f) separating the hybridized labeled single-stranded nucleic acidmolecule from the double-stranded cDNA inserts, thereby generating asubtracted cDNA library of a cell. The invention also provides differentuses of the generated library.

This invention also provides an isolated nucleic acid molecule encodinga protein produced by a melanoma differentiation associated gene. Thisinvention further provides different uses of the isolated malanomadifferentiation genes.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-B Effect of continuous exposure to various growth anddifferentiation modulating agents on the 96-h growth of the humanmelanoma cell line, HO-1. Cells were seeded at 5×10⁴ /35-mm tissueculture plate and 24 h later the medium was changed with the indicatedcompounds. Cell numbers were determined by Coulter Counter after 96-hgrowth. Further details can be found in Materials and Methods. Theabbreviations and the concentrations of test compounds uses: IFN-β+MEZ(recombinant human fibroblast interferon+mezerein)) (2,000 U/ml+10ng/ml); MPA+MEZ (mycophenolic acid+MEZ) (3.0 μM+10 ng/ml); RA+MEZ(trans-retinoic acid+MEZ) (2.5 μM+10 ng/ml); IFN-β+IFN-G(IFN-β+recombinant human gamma interferon) (1,000 U/ml+1,000 U/ml); MEZ(10 ng/ml); MPA (3.0 μM); IFN-G (2,000 U/ml); IFN-β (2,000 U/ml); RA(2.5 μM); and Control (media only).

FIG. 2 Effect of growth and differentiation modulating agents onsteady-state c-jun, jun-B, c-myc, HLA Class I antigen, HLA Class II(DR.sub.β), ISG-54, ISG-15, gro/MGSA, and GAPDH mRNA levels in HO-1cells. Total RNA was isolated 96 h after treatment with the variousagents. The concentrations of compounds used were the same as those usedfor growth studies (see legend to FIG. 1). Ten micrograms of totalcytoplasmic RNA was electrophoresed, transferred to nylon filters, andhybridized with the indicated ³² P-labeled gene probes. Further detailscan be found in Materials and Methods of the first series ofexperiments.

FIG. 3 Effect on gene expression in HO-1 cells of continuous andtransient exposure to agents inducing a reversible commitment todifferentiation or terminal cell differentiation. In the left panel [24h (+)], HO-1 cells were treated with IFN-β+MEZ (2,000 U/ml+10 ng/ml),MPA+MEZ (3.0 μM+10 ng/ml), RA+MEZ (2.5 μM+10 ng/ml), or MEZ (50) (50ng/ml) for 24 h. In the right panel [24 h (+) 72 h (-)], HO-1 cells weretreated with the same test agents used in the left panel for 24 h, thecells were washed three times in serum-free medium and cells werecultured for an additional 72 h in DMEM-10 in the absence of testcompound. Total RNA was isolated, electrophoresed, transferred to nylonfilters, and hybridized with the indicated ³² P-labeled gene probes.Further details can be found in Materials and Methods of the firstseries of experiments.

FIG. 4 Effect of growth and differentiation modulating agents onsteady-state α₅ integrin, β₁ integrin, fibronectin, β-actin, γ-actin,tenascin, and GAPDH mRNA levels in HO-1 cells. Total RNA was isolated 96h after treatment with the various agents. The concentrations ofcompounds used were the same as those used for growth studies (seelegend to FIG. 1). Ten micrograms of total RNA was electrophoresed,transferred to nylon filters, and hybridized with the indicated ³²P-labeled gene probes. Further details can be found in Materials andMethods of the first series of experiments.

FIG. 5 Effect on gene expression in HO-1 cells of continuous andtransient exposure to agents, inducing a reversible commitment todifferentiation or terminal cell differentiation. Experimental detailsare described in the legend to FIG. 3 and in Materials and Methods ofthe first series of experiments.

FIG. 6 Effect of conditioned medium from HO-1 cells treated with agents,resulting in a reversible commitment to differentiation or terminal celldifferentiation on gene expression in naive HO-1 cells. HO-1 cells wereuntreated (Control) or treated for 24 h with IFN-β+MEZ (2,000 U/ml+10ng/ml), MPA+MEZ (3.0 μM+10 ng/ml), RA+MEZ (2.5 μM+10 ng/ml), or MEZ (50)(50 ng/ml). The medium was then removed, the cultures were washed threetimes with serum-free medium, and grown for an additional 72 h incomplete growth medium. The conditioned medium was then collected, andcontaminating cells were removed by centrifugation. Conditioned mediumwas mixed with an equal volume of DMEM-10 (1:2), left panel CONDITIONEDMEDIUM (1:2) (24 h), and applied to previously untreated (naive) HO-1cells for 24 h. Alternatively, conditioned medium was mixed with threeparts of DMEM-10 (1:4), right panel CONDITIONED MEDIUM (1:4) (96 h), andapplied to previously untreated (naive) HO-1 cells for 96 h. Total RNAwas isolated and analyzed by Northern blotting with the DNA probesindicated. Further details can be found in Materials and Methods of thefirst series of experiments.

FIG. 7 Effect of conditioned medium from HO-1 cells treated with agents,resulting in a reversible commitment to differentiation or terminal celldifferentiation on extracellular matrix, extracellular matrix receptor,and cytoskeletal gene expression in naive HO-1 cells. Experimentaldetails are as described in the legend to FIG. 6 and in Materials andMethods of the first series of experiments.

FIG. 8 Flowchart for constructing a subtractive differentiation inducertreated human melanoma cell cDNA library. cDNA libraries wereconstructed from differentiation inducer [IFN-β (2000 units/ml)+MEZ (10ng/ml)] treated human melanoma (HO-1) cells (Ind⁺) and untreated control(Ind⁻) HO-1 cells. Using the protocols outlined, an HO-1 IFN-β+MEZ(Ind⁺) subtracted cDNA library was constructed.

FIG. 9 Determination of the purity of the single-stranded DNA anddouble-stranded DNA-preparations of cDNA libraries. Photograph of a 1%agarose electrophoresis gel stained with ethidium bromide andphotographed under illumination with UV light. The single-stranded DNAwas prepared from the control (Ind⁻) library and double-stranded DNAfrom IFN-β+MEZ (Ind⁺) library. Lane M, λ HindIII molecular weightmarkers; lane 1, single-stranded DNA; lane 2, single-stranded DNAdigested with EcoRI and Xhol; lane 3, double-stranded DNA; lane 4,double-stranded DNA digested with EcoRI and Xhol.

FIG. 10 Northern blot analyses of untreated control and IFN-β, MEZ andIFN-β+MEZ treated HO-1 cells probed with cDNA clones isolated from anHO-1 IFN-β+MEZ (Ind⁺) subtracted cDNA library. RNA was isolated fromcells untreated or treated for 24 hours with IFN-β (2000 units/ml), MEZ(10 ng/ml) or IFN-β+MEZ (2000 units/ml and 10 ng/ml). Ten micrograms oftotal cellular RNA were separated on a 1% agarose gel, transferred tonylon membranes and then hybridized individually with radioactive probesprepared using appropriate inserts from the cDNA clones. The series ofcDNAs isolated from inducer treated HO-1 cells have been tentativelycalled mda, melanoma differentiation associated, genes, Glyceraldehydephosphate dehydrogenase (GAPDH) was used as a control for uniformloading and expression of the RNA samples.

FIG. 11 Homology of mda-3 to the cDNA of human macrophage inflammatoryprotein (GOS19-1). The DNA sequence of mda-3 was obtained using theSanger dideoxynucleotide sequencing method. For sequence homology withknown genes, the DNA sequence of mda-3 was compared using the GCG/FASTAprogram and the GenBank/EMBL database. The top sequence corresponds tothe mda-3 cDNA and the bottom sequence corresponds to GOS19-1.

FIG. 12 Effect of IFN-β and MEZ, alone and in combination on the growthof HO-1 human melanoma cells (see third series of experiments forfurther details).

FIG. 13 Effect of IFN-β and MEZ, alone and in combination on themorphology of HO-1 human melanoma cells. Cells were treated with 2,000units/ml of IFN-β, 10 ng/ml MEZ or the combination of agents for 24 h.

FIG. 14 Expression of proliferative sensitive proteins (P2Ps) in humanmelanoma cells treated with growth suppressing and differentiationinducing compounds. The combination of IFN-β+MEZ induces terminaldifferentiation in FO-1 human melanoma cells, but not inSV40-transformed human melanocytes. Experimental details for determiningP2Ps can be found in Minoo et al. (52) and Witte and Scott (53) of thethird series of experiments.

FIG. 15 Northern blot analyses of untreated and treated HO-1 cellsprobed with cDNA clones isolated from an HO-1 IFN-β+MEZ (Ind⁺)subtracted cDNA library. RNA was isolated from cells treated for 96 hwith the agents indicated. Concentration of agents were the same as usedin FIG. 1. Experimental details can be found in Jiang and Fisher (49)and Jiang et al. (14) of the third series of experiments. IFN-β 2,000units/ml; IFN-λ 2,000 units/ml; MEZ 10 ng/ml; MPA 3 μM; RA 2.5 μM;IFN-β+IFN-γ (1,000 units/ml of each IFN); IFN-β+MEZ (2,000 units/ml+10ng/ml); MPA+MEZ (3 μM+10 ng/ml); RA+MEZ (2.5 μM+10 ng/ml).

FIG. 16 Expression of mda genes in human melanomas. -=control,+=IFN-β+MEZ (2,000 units/ml±10 ng/ml).

FIG. 17 Expression of mda genes in normal cerebellum, glioblastomamultiforme and normal skin fibroblasts. -=control, +=IFN-β+MEZ (2,000units/ml+10 ng/ml).

FIG. 18 Expression of mda genes in human colorectal carcinoma (SW613),endometrial adenocarcinoma (HTB113) and prostate carcinoma (LNCaP).-=control, +=IFN-β+MEZ (2,000 units/ml+10 ng/ml).

FIG. 19 Effect of various treatment protocols on mda expression in HO-1cells. IFN-β (2,000 units/ml, 24 hours); MEZ (10 ng/ml, 24 hours);IFN-β+MEZ (2,000 units/ml+10 ng/ml, 24 hours); phenyl butyrate (PB) (4mM, 24 hours, 4d, 7d); γ Rad (γ irradiation) 3 gray, 24 hours, Act D(actinomycin D) (5 μg/ml, 2 hour→24 hours assay); Adriamycin (Adr) (0.1μg/ml, 24 hours); Vincristine (Vin) (0.1 μg/ml, 24 hours); cis-plt(cis-platinum) (0.1 μg/ml, 24 hours); TNF-α (tumor necrosis factor-α)(100 units/ml, 24 hours); UV (10 joules/mm², 2, 14 and 24 hours assay);VP-16 (5 μg/ml, 24 hours); IFN-α (2,000 units/ml, 24 hours); andIFN-α+MEZ (2,000 units/ml+10 ng/ml, 24 hours).

FIG. 20. Stages in the development of metastatic melanoma.

FIGS. 21A-H. Effect of recombinant human IFN-β and mezerein (MEZ) usedalone and in combination on the morphology of HO-1 and BO-2 humanmelanoma cells (X150). (A) Control HO-1 cells 24 h postplating. (B) HO-1cells exposed to 2,000 units/ml IFN-β for 24 h. (C) HO-1 cells exposedto 10 ng/ml MEZ for 24 h. (D) HO-1 cells exposed to 2,000 units/ml IFN-βand 10 ng/ml MEZ for 24 h. (E) Control BO-2 cells 24 h postplating. (F)BO-2 cells exposed to 2,000 units/ml IFN-β for 24 h. (G) BO-2 cellsexposed to 10 ng/ml MEZ for 24 h. (H) BO-2 cells exposed to 2000units/ml IFN-β and 10 ng/ml-MEZ for 24 h. Data from reference [18] ofthe fifth series of experiments.

FIG. 22 Components of the differentiation process in HO-1 human melanomacells. Abbreviations: IFN-β: recombinant human fibroblast interferon;IFN-γ: recombinant immune interferon; MPA: mycophenolic acid; MEZ:mezerein; Trans RA: trans retinoic acid. Relative growth suppression:4+=˜80% reduction in growth in comparison with untreated controlcultures; 3+=˜50 to 60% reduction in growth in comparison with untreatedcontrol cultures; 2+=˜40% reduction in growth in comparison withuntreated control cultures; 1+=˜30% reduction in growth comparison withuntreated control cultures.

FIGS. 23A-B cDNA and predicted amino acid sequence of mda-6. Thepredicted translation begins at nucleotide 95 and ends at nucleotide589. Accession number U09579 (GeneBank).

FIG. 24 Cellular changes mediating enhanced expression of certain mdagenes and differential expression of particular mda genes during tumorprogression and in normal versus tumor-derived cell types. Specific mdagenes have been identified that display enhanced expression duringtreatment with agents that induce growth suppression, DNA damage(including chemotherapeutic agents that function by differentmechanisms) and/or terminal differentiation. Additional mda genes havealso been shown to display differential expression as a function oftumor progression and in matched sets of normal versus tumor-derivedhuman cells.

FIGS. 25A-B Induction of mda-6 (WAF1/CIP1/SDI1) expression in HL-60cells by TPA and RA. Total cytoplasmic RNA was isolated from HL-60 cellstreated with TPA (3 nM) for 0.25, 0.5, 1, 2, 4 or 6 d and from HL-60cells treated with RA (1 μM) for 0.5, 1, 2, 4 or 6 d. A 10-μg aliquot ofRNA was run on a 1.0% agarose gel and transferred to a nylon filter.Blots were hybridized with a multi-prime ³² P-labeled mda-6 gene probe.Filters were stripped and rehybridized with a multiprime ³² P-labeledGAPDH probe.

FIGS. 26A-C Early induction of mda-6 (WAF1/CIP1/SDI1) expression inHL-60 and TPA-resistant HL-60 (HL-525) cells treated with TPA, RA andVit D3. Cells were treated with TPA (3 nM), RA (1 μM) or Vit D3 (400 nM)for 1, 2, 3, 6 or 12 h. RNA was isolated and analyzed by RT-PCR usingappropriate mda-6 or GAPDH specific primers as described in theMaterials and methods.

FIGS. 27A-C Induction of mda-6 (WAF1/CIP1/SDI1) expression in HL-60 andTPA-resistant HL-60 (HL-525) cells after extended incubation with TPA,RA and Vit D3. Cells were treated with TPA (3 nM), RA (1 μM) or Vit D3(400 nM) for 0.5, 1, 2, 4 or 6 d. RNA was isolated and analyzed byRT-PCR using appropriate mda-6 or GAPDH specific primers as described inthe Materials and methods.

FIG. 28 Induction of the MDA-6 (WAF1/CIP1/SDI1) encoded protein p21 inHL-60 cells treated with TPA, DMSO and RA. Lysates from untreated HL-60(control) and HL-60 cells treated with TPA (3 nM), DMSO (1%) or RA (1μM) for 12, 24, 48 and 72 h were immunoprecipitated with WAF1/CIP1(MDA-6) polyclonal antibody or actin monoclonal antibody as described inMaterials and methods. The size of the MDA-6 protein is 21 kDa and thesize of the Actin protein is 42 kDa.

FIGS. 29A-B Effect CHX on the induction of mda-6 in HL-60 andTPA-resistant HL-60 (HL-525) cells bells were treated with CHX (10μg/ml) for 1, 3, 6 or 10 h or with CHX (10 μg/ml) plus TPA (3 nM) for1+, 3+, 6+ or 10+ h. RNA was isolated and analyzed by RT-PCR usingappropriate mda-6 or GAPDH specific primers as described in theMaterials and methods in the sixth series of experiments.

FIG. 30 Open-reading frame of mda-6. Predicted translation of the mda-6cDNA begins at nucleotide 95 and ends a nucleotide 589. Accession numberU09579 (Genbank). mda-6 encodes a 164 amino acid protein with an M of21,000 that is identical to the cyclin dependent kinase inhibitor, p21.

FIGS. 31A-E Induction of mda-6 (p21) expression in HO-1 human melanomacells as a function of differentiation and growth suppression. HO-1cells were untreated (control) or treated for 24 h (panel A), 96 h(panel B) or treated for 24 h followed by growth for 72 h in the absenceof inducer (panel C) with IFN-β (2000 units/ml), MEZ (10 ng/ml) orIFN-β+MEZ (2000 units/ml+10 ng/ml). In panel D, HO-1 cells were grownfor the indicated time in medium containing 5% fetal bovine serum(Control) or without fetal bovine serum (DMEM-0). In panel E,high-density HO-1 cells were incubated in DMEM-0 for the timesindicated. RNA isolation, Northern blotting and hybridization with mda-6and GAPDH was performed as described (Jiang & Fisher, 1993; Jiang etal., 1993).

FIG. 32 Effect of IFN-β+MEZ on mda-6 (p21) expression in human melanomacells and an SV40-transformed human melanocyte culture. High-densitymelanoma cells (HO-1, FO-1, LO-1, SH-1, WM239 and WM278) andlogarithmically growing low-density SV40-transformed human melanocytes(SV516-SV) were treated with IFN-β (2000 units/ml)+MEZ (10 ng/ml) for 24hr, total RNA was isolated and analyzed by Northern blotting (Jiang &Fisher, 1993; Jiang et al., 1993). Filters were probed with mda-6 thenstripped and probed with GAPDH as described (Jiang & Fisher, 1993; Jianget al., 1993).

FIG. 33 Effect of 24 h treatment of HO-1 cells with IFN-β, MEZ andIFN-β+MEZ on p53 and p21 levels. Lysates from untreated (control) andHO-1 cells treated with IFN-β (2000 units/ml), MEZ (10 ng/ml) orIFN-β+MEZ (2000 units/ml+10 ng/ml) for 24 h were immunoprecipitated withp53 monoclonal antibody Ab1 (PAb421), p21 (WAF1/CIP1) polyclonalantibody or an actin monoclonal antibody as described in Materials andmethods in the seventh series of experiments.

FIG. 34 Expression of p53 and p21 as a function of growth arrest andterminal differentiation in HO-1 cells. Lysates from untreated (control)and HO-1 cells treated with IFN-β (2000 units/ml), MEZ (10 ng/ml) orIFN-β+MEZ (2000 units/ml+10 ng/ml) for 48, 72 and 96 h wereimmunoprecipitated with p53 monoclonal antibody Ab1 (PAb421), p21(WAF1/CIP1) polyclonal antibody or an actin monoclonal antibody asdescribed in Materials and methods in the seventh series of experiments.

FIG. 35 Relative expression of mda-6 during melanoma progression. Thelevel of mda-6 relative to GAPDH was determined by quantitative(comparative) RT-PCR in actively growing cells. RGP: radial growth phaseprimary melanoma. VGP: vertical growth phase primary melanoma. Resultsrepresent average for: 5 melanocyte cultures, one dysplastic nevusculture, one SV40-transformed immortalized melanocyte culture, one RGPand four early VGP primary melanomas and six metastatic melanomas.

FIG. 36 Expression of mda-6 (p21) as a function of Matrigel-mediatedprogression in primary human melanomas. The level of mda-6 and GAPDH wasdetermined by RT-PCR for the indicated actively proliferating celllines. P1 and P2 refers to the first and second passage, respectively,through nude mice of RGP or early VGP melanoma cells in combination withMatrigel.

FIG. 37 Northern blot analysis of mda-6 (p21) expression in C8161 humanmelanoma and chromosome 6 containing C8161 human melanoma subclones.Levels of mda-6 and GAPDH mRNAs were determined in actively growinguntreated cells (-) and cells treated for 96 hr with IFN-β+MEZ (1000units/ml+10 ng/ml) as described (Jiang & Fisher, 1993; Jiang et al.,1994a).

FIGS. 38A-B Nucleotide sequence and deduced encoded amino acid sequenceof the mda-7 cDNA. The hydrophobic putative transmembrane domain andthree potential N-glycosylation residues are underlined; the 3'untranslated sequence contains three putative instability motifs ATTA(SEQ ID NO:16) (underlined) and two consensus signals AATAAA (SEQ IDNO:17) for poly(A) addition (underlined).

FIG. 39 Properties and structure of the mda-7 encoded gene product aspredicted by GCG/Plotstructure program.

FIG. 40 Hydrophobic analysis and structural predictions of the mda-7encoded gene product by GCG/Pepplot program. A potential transmembranedomain is predicted using this program.

FIG. 41 Structure of mda-7 encoded gene produce as predicted by theChou-Fosman method.

FIG. 42 Relative expression of mda-7 during melanoma progression. Thelevel of mda-7 relative to GAPDH was determined quantitative(comparative) RT-PCR in actively growing cells. The cell lines examinedwere (1) normal melanocytes; (2) primary melanoma cells, including RGPand VGP cells; and (3) metastatic melanoma cells.

FIG. 43 Expression of mda-6 and mda-7 as a function of aging andsenescence in human fibroblast cells. IMR90 cells analyzed at latepassage (OLD) versus early passage (YOUNG). IDH4 cells are IMR90 cellsimmortalized by an SV40 T-antigen (transcriptionally controlled by adexamethasone (DEX) inducible mouse mammary tumor virus promoter).Growth of IDH4 cells in DEX results in active growth and expression ofan immortalized phenotype (a YOUNG IMR90 phenotype). In contrast,removal of DEX results in a shutdown of the T-antigen and loss ofproliferative capacity and senescence (SENESCENT). RNA was extractedfrom the four cell lines and analyzed by reversetranscription-polymerase chain reaction (RT-PCR) using primer sequencesspecific for mda-6, mda-7 or GAPDH. As a control for expression of thesegenes, RNA from HO-1 human melanoma cells treated with IFN-β+MEZ (2000units/ml+10 ng/ml) for 96 hr was used. HO-1 cells under these conditionsare irreversibly growth arrested and terminally differentiated.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this application, the following standard abbreviations areused to indicate specific nucleotides:

                  TABLE 1                                                         ______________________________________                                        Experimental                                                                             Morphology   Melanin  Tyrosinase                                     Conditions.sup.a changes.sup.b Synthesis.sup.c activity.sup.d               ______________________________________                                          RA (2.5 μM) - 1+ 2+                                                        MPA (3.0 μM) + 2+ 3+                                                       MEZ + 1+ NT                                                                   (10 ng/ml)                                                                    IFN-β - 1+ NT                                                            (2000 U/ml)                                                                   IFN-γ - - NT                                                            (2000 U/ml)                                                                   RA + MEZ + NT NT                                                              (2.5 μM +                                                                  10 ng/ml)                                                                     MPA + MEZ + NT NT                                                             (3.0 μM +                                                                  10 ng/ml)                                                                     IFN-β + IFN-γ - 1+ NT                                              (1000 U/ml +                                                                  1000 U/ml)                                                                    IFN-β + MEZ  + 4+ NT                                                     (2000 U/ml +                                                                  10 ng/ml)                                                                   ______________________________________                                                      Growth                                                            Experimental Suppression Terminal cell                                        conditions.sup.a (reversible).sup.e differentiation.sup.f                   ______________________________________                                          RA (2.5 μM) - -                                                            MPA (3.0 μM) 3+ -                                                          MEZ 1+ -                                                                      (10 ng/ml)                                                                    IFN-β 3+ -                                                               (2000 U/ml)                                                                   IFN-γ 2+ -                                                              (2000 U/ml)                                                                   RA + MEZ 1+ -                                                                 (2.5 μM +                                                                  10 ng/ml)                                                                     MPA + MEZ 3+ -                                                                (3.0 μM +                                                                  10 ng/ml)                                                                     IFN-β + IFN-γ                                                      (1000 U/ml +                                                                  1000 U/ml)                                                                    IFN-β + MEZ  4+.sup.g +                                                  (2000 U/ml +                                                                  10 ng/ml)                                                                   ______________________________________                                         .sup.a HO1 cells were grown for 96 hr or for 6 or 7 days (with medium         changes after 3 or 4 days) in the presence of the agents indicated. For       morphology, cells grown for 96 hr in the test agent were observed             microscopically. For melanin synthesis, results are for 6d assays for RA      and MPA (11) or 7d assays for MEZ, IFNβ, IFNγ, IFNβ +         IFNγ and IFNβ + MEZ (6,12). For tyrosinase assays, results are     for 6d assays for RA, MPA and MEZ (10). Growth  # suppression (reversible     and terminal cell differentiation) assays, refer to cultures treated with     the indicated compound(s) for 96 hr prior to cell number determination, o     treated for 96 hr and then grown for 2 weeks (with medium changes every 4     days) in the absence of compound prior to cell number determination.          .sup.b Morphology changes refer to the development of dendritelike            processes 96 hr after growth in the indicated compound. + = presence of       dendritelike processes; - = no dendritelike processes.                        .sup.c Melanin assays were determined as described in refs. 6,8,11,12.        Results are expressed as relative increases based on separate data            presented in refs. 6,8,11,12. N.T. = not tested.                              .sup.d Tyrosinase assays were performed as described in ref. 11. Relative     increases (of a similar magnitude) were found for RA, MPA and MEZ after 6     days exposure to these agents (11). N.T. = not tested.                        .sup.e Reversible growth suppression indicates resumption of cell growth      after treatment with the indicated compound(s) for 96 hr, removal of the      test agent and growth for 14 days in compound(s) free medium. Further         details can be found in materials and methods. The degree of initial 96 h     growth suppression is indicated as: - = no significant change in growth       (<10% reduction in growth in comparison with untreated control cultures);     1+  # ˜30% reduction in growth in comparison with untreated control     cultures; 2+ = ˜40% reduction in growth in comparison with untreate     control cultures; 3+ = ˜50 to 60% reduction in growth in comparison     with untreated control cultures; 4+ = ˜80% reduction in growth in       comparison with untreated control cultures.                                   .sup.f The combination of IFNβ + MEZ results in irreversible growth      suppression.                                                                  .sup.g The combination of IFNβ + MEZ results in irreversible growth      suppression                                                              

This invention provides a method of generating a subtracted cDNA libraryof a cell comprising: a) generating a cDNA library of the cell; b)isolating double-stranded DNAs from the cDNA library; c) releasing thedouble-stranded cDNA inserts from the double-stranded DNAs; d)denaturing the isolated double-stranded cDNA inserts; e) hybridizing thedenatured double-stranded cDNA inserts with a labelled single-strandednucleic acid molecules which are to be subtracted from the cDNA library;and f) separating the hybridized labeled single-stranded nucleic acidmolecule from the double-stranded cDNA inserts, thereby generating asubtracted cDNA library of a cell.

In an embodiment, the cDNA library of the cell is a λZAP cDNA library.

This invention provides the above-described method, wherein thereleasing of the double-stranded cDNA is performed by digestion withappropriate restriction enzymes.

This invention also provides the above-method of generating a subtractedcDNA library of a cell, wherein the denaturing of step d) is by boiling.

In an embodiment, the single-stranded nucleic acid molecules are DNAs.In a further embodiment, the single-stranded nucleic acid molecules arelabelled with biotin. In a still further embodiment, the single-strandednucleic acid molecules are labelled with biotin, wherein the separatingof step f) is performed by extraction with streptavidin-phenol:Chloroform.

Other methods for labelling single-stranded nucleic acid molecules arewell-known in the art.

This invention further provides the above-described methods ofgenerating a subtracted cDNA library of a cell, wherein thesingle-stranded nucleic acid molecules are from another cDNA library. Inanother embodiment, this cDNA library is a λZAP cDNA library.

In another embodiment, the single-stranded nucleic acid molecules arefrom another cDNA library, wherein the cDNA library is a λZAP cDNAlibrary, wherein the cell is an HO-1 melanoma cell treated with IFN-βand MEZ.

In another embodiment, wherein the cDNA library is a λZAP cDNA library,the cell is an HO-1 melanoma cell treated with IFN-β and MEZ and thesingle-stranded nucleic acid molecules are from another cDNA library ofa HO-1 melanoma cell.

In still another embodiment of the above-described methods of generatinga subtracted cDNA library of a cell, wherein the cDNA library is a λZAPcDNA library, wherein the cell is terminally differentiated and thesingle-stranded nucleic acid molecules are from another cDNA library ofan undifferentiated cell.

In still another embodiment of the above-described methods of generatinga subtracted cDNA library of a cell, wherein the cDNA library is a λZAPcDNA library, wherein the cell is undifferentiated and thesingle-stranded nucleic acid molecules are from another cDNA library ofa terminally differentiated cell.

In one embodiment of the above-described method of generating asubtracted cDNA library of a cell, the cell is selected from a groupconsisting essentially of neuroblastoma cell, glioblastoma multiformecell, myeloid leukemic cell, breast carcinoma cell, colon carcinomacell, endometrial carcinoma cell, lung carcinoma cell, ovarian carcinomacell and prostate carcinoma cell.

In another embodiment of the above-described method of generating asubtracted cDNA library of a cell, the cell is induced to undergoreversible growth arrest or DNA damage and the single-stranded nucleicacid molecules are from another cDNA library of an uninduced cell.

In still another embodiment of the above-described method of generatinga subtracted cDNA library of a cell, the cell is at one developmentalstage and the single-stranded nucleic acid molecules are from anothercDNA library from the cell at different developmental stage.

In still another embodiment of the above-described method of generatinga subtracted cDNA library of a cell, the cell is cancerous and thesingle-stranded nucleic acid molecules are from another cDNA libraryfrom a normal cell.

In another embodiment, the cell is from the breast, brain, meninges,spinal cord, colon, endometrium, lung, prostate and ovary.

This invention also provides a method of generating a subtracted cDNAlibrary of a cell comprising: a) generating a cDNA library of the cell;b) isolating double-stranded DNAs from the cDNA library; c) releasingthe double-stranded cDNA inserts from the double-stranded DNAs; d)denaturing the isolated double-stranded cDNA inserts; e) hybridizing thedenatured double-stranded cDNA inserts with a labelled single-strandednucleic acid molecules which are to be subtracted from the cDNA library;and f) separating the hybridized labeled single-stranded nucleic acidmolecule from the double-stranded cDNA inserts, thereby generating asubtracted cDNA library of a cell, wherein the single-stranded nucleicacid molecules are from another cDNA library, wherein the cDNA libraryis a λZAP cDNA library, further comprising introducing the subtractedlibrary into host cells.

This invention provides a subtracted library generated by the methodgenerating a subtracted cDNA library of a cell comprising: a) generatinga cDNA library of the cell; b) isolating double-stranded DNAs from thecDNA library; c) releasing the double-stranded cDNA inserts from thedouble-stranded DNAs; d) denaturing the isolated double-stranded cDNAinserts; e) hybridizing the denatured double-stranded cDNA inserts witha labelled single-stranded nucleic acid molecules which are to besubtracted from the cDNA library; and f) separating the hybridizedlabeled single-stranded nucleic acid molecule from the double-strandedcDNA inserts, thereby generating a subtracted cDNA library of a cell.

This invention provides a subtracted library generated by the method ofgenerating a subtracted cDNA library of a cell comprising: a) generatinga cDNA library of the cell; b) isolating double-stranded DNAs from thecDNA library; c) releasing the double-stranded cDNA inserts from thedouble-stranded DNAs; d) denaturing the isolated double-stranded cDNAinserts; e) hybridizing the denatured double-stranded cDNA inserts witha labelled single-stranded nucleic acid molecules which are to besubtracted from the cDNA library; and f) separating the hybridizedlabeled single-stranded nucleic acid molecule from the double-strandedcDNA inserts, thereby generating a subtracted cDNA library of a cell,wherein the single-stranded nucleic acid molecules are from another cDNAlibrary, wherein the cDNA library is a λZAP cDNA library, wherein thecell is an HO-1 melanoma cell treated with IFN-β and MEZ, wherein thesingle-stranded nucleic acid molecules are from another cDNA library ofa HO-1 melanoma cell.

This invention provides a method of identifying a melanomadifferentiation associated gene comprising: a) generating probes fromclones of the above-described subtracted library; and b) hybridizing theprobe with total RNAs or mRNAs from HO-1 cells treated with IFN-β andMEZ and total RNAs or mRNAs from untreated HO-1 cells, hybridization ofthe probe with the mRNAs from the treated HO-1 cell but no or reducedhybridization with the total RNAs or mRNAs from untreated cellsindicating that the clone from which the probe is generated carries amelanoma differentiation associated gene.

This invention further provides a melanoma differentiation associatedgene identified by the above method.

This invention provides a nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence of mda-4gene.

This invention further provides a method of detecting the expression ofmda-4 gene in a cell comprising: a) isolating the nucleic acids in thecell; b) hybridizing the isolated nucleic acids with the nucleic acidmolecule capable of specifically hybridizing with a sequence of mda-4gene under conditions permitting hybrid formation; and c) detectinghybrids formed, the detection of the hybrids indicating expression ofmda-4 gene in the cell.

This invention also provides a method to indicate the tissue lineage ofa cell comprising detecting the expression of mda-4 gene using theabove-described method, the expression of mda-4 gene indicating that thetissue lineage of the cell is neuroectodermal.

This invention also provides a method for distinguishing a fibroblast orepithelial cell from a melanoma or central nervous system cellcomprising detecting the expression of mda-4 gene using theabove-described method, the expression of mda-4 indicating that the cellis a melanoma cell or a central nervous system lineage cell.

This invention provides a method to monitor the response to DNA damageinduced by gamma irradiation and UV irradiation of a cell comprisinghybridizing the nucleic acid molecule capable of specificallyhybridizing with a sequence of mda-4 gene, hybridization of the nucleicacids from the cell indicating that there is a response to the DNAdamage of the cell.

This invention provides a method of monitoring a response to treatmentwith chemotherapeutic agents which work in a similar manner ascis-platinum in a cell comprising hybridizing the nucleic acids from thecell with the nucleic acid molecule capable of specifically hybridizingwith a sequence of mda-4 gene, hybridization of the nucleic acids fromthe cell responds to the treatment with the chemotherapeutic agents.

This invention further provides a method for detecting types I or IIinterferons in a sample comprising: a) incubating the sample with the 5'regulatory element of the mda-4 gene under conditions permitting bindingof types I and II interferons transcriptional regulatory proteins to theregulatory elements; and b) detecting the binding of types I or IIinterferons transcriptional regulatory proteins to the regulatoryelements, the binding indicating the presence of type I or IIinterferons in the sample.

As used herein, the term "sample" is broadly defined. It includes, butnot limited to bodily fluids such as urine, saliva, blood and otherclinical samples.

Transcriptional regulatory proteins which are responsive to type I or IIinterferons are well-known in the art.

In an embodiment, the target cell is a eukaryotic cell. In a separateembodiment, the 5' regulatory element is linked to the native mda-4 geneand the detection of binding is by examination of the elevatedexpression of the mda-4 gene. In an embodiment, the 5' regulatoryelement is linked to a marker gene. In a further embodiment, the markergene is β-galactosidase or CAT.

This invention provides an isolated nucleic acid molecule encoding aprotein produced by a melanoma differentiation associated genedesignated mda-1. In an embodiment, the nucleic acid is a cDNA. Inanother embodiment, the nucleic acid is genomic DNA.

In a separate embodiment, the mda-1 gene is coding for a human protein.

This invention provides an isolated nucleic acid molecule encoding aprotein produced by a melanoma differentiation associated genedesignated mda-2. In an embodiment, the nucleic acid is a cDNA. Inanother embodiment, the nucleic acid is genomic DNA.

In a separate embodiment, the mda-2 gene is coding for a human protein.

This invention provides an isolated nucleic acid molecule encoding aprotein produced by a melanoma differentiation associated genedesignated mda-4. In an embodiment, the nucleic acid is a cDNA. Inanother embodiment, the nucleic acid is genomic DNA.

In a separate embodiment, the mda-4 gene is coding for a human protein.

This invention provides an isolated nucleic acid molecule encoding aprotein produced by a melanoma differentiation associated genedesignated mda-5. In an embodiment, the nucleic acid is a cDNA. Inanother embodiment, the nucleic acid is genomic DNA.

In a separate embodiment, the mda-5 gene is coding for a human protein.

This invention provides a nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence of thenucleic acid molecule, mda-5.

This invention provides a method of detecting the expression of mda-5gene in a cell comprising: a) isolating the nucleic acids in the cell;b) hybridizing the isolated nucleic acids with the nucleic acidmolecules of at least 15 nucleotides capable of specifically hybridizingwith a sequence of the nucleic acid molecule mda-5 under conditionspermitting hybrids formation; and c) detecting hybrids formed, thedetection of the hybrids formed indicating the expression of mda-5 genein the cell.

This invention provides a method for distinguishing a normalneuroectodermal cell from a malignant neuroectodermal cell comprisingdetecting the expression of mda-5 gene using the method of detecting theexpression of mda-5 gene in a cell comprising: a) isolating the nucleicacids in the cell; b) hybridizing the isolated nucleic acids with thenucleic acid molecules of at least 15 nucleotides capable ofspecifically hybridizing with a sequence of the nucleic acid moleculemda-5 under conditions permitting hybrids formation; and c) detectinghybrids formed, the detection of the hybrids formed indicating theexpression of mda-5 gene in the cell, the expression of mda-5 geneindicating that the cell is normal neuroectodermal cell.

This invention provides a method for detecting types I or II interferonsin a sample comprising: a) incubating the sample with the 5' regulatoryelement of the mda-5 gene under conditions permitting binding of types Iand II interferons transcriptional regulatory proteins to the regulatoryelements; and b) detecting the binding of the types I or II interferonstranscriptional regulatory proteins to the regulatory elements, thebinding indicating the presence of type I or type II interferons in thesample.

In an embodiment, the cell is a eukaryotic cell. In another embodiment,the 5' regulatory element is linked to the native mda-5 gene and thedetection of binding is by the examination of the elevated expression ofmda-5 gene.

In a separate embodiment, the 5' regulatory element is linked to amarker gene. In a further embodiment, the marker gene isβ-galactosidase, luciferase or CAT.

This invention provides a method for identifying a compound capable ofinducing terminal differentiation in human melanoma cells comprising: a)incubating the human melanoma cells with an appropriate concentration ofthe compound; and b) detecting the expression of mda-5 using theabove-described method, the expression of mda-5 gene indicating that thecompound is capable of inducing terminal differentiation in humanmelanoma cells.

This invention provides an isolated nucleic acid molecule encoding aprotein produced by a melanoma differentiation associated genedesignated mda-6. In an embodiment, the nucleic acid is a cDNA. Inanother embodiment, the nucleic acid is genomic DNA.

In a separate embodiment, the mda-6 gene is coding for a human protein.

This invention provides a nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence of thenucleic acid molecule, an isolated nucleic acid molecule encoding aprotein produced by a melanoma differentiation associated genedesignated mda-6.

This invention provides a method of detecting the expression of mda-6gene in a cell comprising: a) isolating the nucleic acids in the cell;b) hybridizing the isolated nucleic acids with the nucleic acidmolecules of a nucleic acid molecule of at least 15 nucleotides capableof specifically hybridizing with a sequence of the nucleic acidmolecule, an isolated nucleic acid molecule encoding a protein producedby a melanoma differentiation associated gene designated mda-6 underconditions permitting hybrids formation; and c) detecting hybridsformed, the detection of the hybrids formed indicating the expression ofmda-6 gene in the cell.

This invention provides a method for distinguishing a normalneuroectodermal cell from a malignant neuroectodermal cell comprisingdetecting the expression of mda-6 gene using the method of detecting theexpression of mda-6 gene in a cell comprising: a) isolating the nucleicacids in the cell; b) hybridizing the isolated nucleic acids with thenucleic acid molecules capable of recognizing the mda-6 gene underconditions permitting hybrids formation; and c) detecting hybridsformed, the detection of the hybrids formed indicating the expression ofmda-6 gene in the cell, the expression of mda-6 gene indicating that thecell is normal neuroectodermal cell.

The invention also provides a method for distinguishing anadenocarcinoma cell from a carcinoma cell comprising detecting theexpression of mda-6 gene using the above-described method, theexpression of mda-6 gene indicating that the cell is a carcinoma cell.

The invention further provides a method for monitoring the response of acell to an anticancer agent such as actinomycin-D or adriamycincomprising detecting the expression of mda-6 gene using theabove-described method, the expression of mda-6 gene indicating that thecell responds to the anticancer agent.

This invention provides a method for monitoring response totopoisomerase inhibitor by a cell comprising detecting the expression ofmda-6 using the above-described method of detecting the expression ofmda-6 gene in a cell, the expression of mda-6 indicating that the cellresponds to the topoisomerase inhibitor.

This invention provides a method for identifying a compound capable ofinducing terminal differentiation in human melanoma cells comprising: a)incubating the human melanoma cells with an appropriate concentration ofthe compound; and b) detecting the expression of mda-6 in a cell usingthe above-described method, the expression of mda-6 gene indicating thatthe compound is capable of inducing terminal differentiation in humanmelanoma cells.

This invention provides a method for identifying a compound capable ofinducing terminal differentiation in human leukemia cells comprising a)incubating the human leukemia cells with an appropriate concentration ofthe compound; and b) detecting the expression of mda-6 using theabove-described method, the expression of mda-6 gene indicating that thecompound is capable of inducing terminal differentiation in humanleukemia cells.

This invention also provides a method for identifying a compound capableof inducing terminal differentiation in human lymphoma cells comprising:a) incubating the human lymphoma cells with an appropriate concentrationof the compound; and b) detecting the expression of mda-6 using theabove-described method, the expression of mda-6 gene indicating that thecompound is capable of inducing terminal differentiation in humanlymphoma cells.

This invention provides a method for identifying a compound capable ofinducing terminal differentiation in human neuroblastoma cellscomprising: a) incubating the human neuroblastoma cells with anappropriate concentration of the compound; and b) detecting theexpression of mda-6 using the above-described method, the expression ofmda-6 gene indicating that the compound is capable of inducing terminaldifferentiation in human neuroblastoma cells.

This invention also provides a method for identifying a compound capableof inducing terminal differentiation in human glioblastoma multiformecells comprising: a) incubating the human glioblastoma cells with anappropriate concentration of the compound; and b) detecting theexpression of mda-6 using the above-described method, the expression ofmda-6 gene indicating that the compound is capable of inducing terminaldifferentiation in human glioblastoma multiforme cells.

This invention also provides a method for distinguishing an early stagefrom a more progressed human melanoma cell comprising detecting theexpression of mda-6 gene using the above-described method, theexpression of mda-6 gene indicating that the cell is a less progressedhuman melanoma cell.

This invention provides a method for reversing the malignant phenotypeof cells comprising: (a) linking the mda-6 gene to a regulatory elementsuch that the expression of the mda-6 gene is under the control of theregulatory element; and (b) introducing the linked mda-6 gene into themalignant cells for the expression of the mda-6 gene, thereby reversingthe malignant phenotype of cells.

This invention also provides a method for reversing the malignantphenotype of cells comprising: (a) linking the mda-6 gene to aregulatory element such that the expression of the mda-6 gene is underthe control of the regulatory element; (b) introducing the linked mda-6gene into the malignant cells; and (c) placing the cells from step (b)in appropriate conditions to express the mda-6 gene such that theexpression of the mda-6 gene will reverse the transforming phenotype ofthe malignant cells.

This invention also provides a method of reversing the phenotype ofmalignant cells in a subject comprising: (a) linking the mda-6 gene to aregulatory element such that the expression of the mda-6 gene is underthe control of the regulatory element; (b) introducing the linked mda-6gene into the malignant cells for the expression of the mda-6 gene,thereby reversing the phenotype of the malignant cells.

This invention also provides a method of reversing the phenotype ofmalignant cells in a subject comprising: (a) linking the mda-6 gene to aregulatory element such that the expression of the mda-6 gene is underthe control of the regulatory element; (b) introducing the linked mda-6gene into the malignant cells of the subject; and (c) inducing theexpression of the mda-6 gene which will reverse the transformingproperties of the cells, thereby reversing the phenotype of themalignant cells in the subject.

This invention also provides a method of inducing growth suppression intumorigenic and metastatic cells comprising: (a) linking the mda-6 geneto a regulatory element such that the expression of the mda-6 gene isunder the control of the regulatory element; (b) introducing the linkedmda-6 gene into the tumorigenic and metastatic cells; and (c) inducingthe expression of the mda-6 gene, thereby inducing growth suppression intumorigenic and metastatic cells.

This invention also provides a method of inducing terminaldifferentiation in tumorigenic and metastatic cells comprising: (a)linking the mda-6 gene to a regulatory element such that the expressionof the mda-6 gene is under the control of the regulatory element; (b)introducing the linked mda-6 gene into the tumorigenic and metastaticcells; and (c) inducing the expression of the mda-6 gene, therebyinducing terminal differentiation in tumorigenic and metastatic cells.

In an embodiment, the cell is a melanoma, leukemia, lymphoma,neuroblastoma, glioblastoma or carcinoma cell.

In a separate embodiment, the regulatory element is a promoter. In afurther embodiment, the promoter is a tissue-specific promoter. Inanother embodiment, the promoter is an inducible promoter.

The linked mda-6 gene may be introduced into the cells by naked DNAtechnology, retroviral vectors, antibody-coated liposomes, mechanical orelectrical means. These technologies are known in the art.

This invention provides a method of determining the stage of a melanomacomprising: (a) obtaining appropriate amount of cells from the melanoma;(b) measuring the expression level of the mda-6 gene in the cells; and(b) comparing the expression level with predetermined standards ofnormal and melanoma cells in different stages, thereby determining thestage of a melanoma.

In an embodiment, the expression is measured by the antibodies againstthe mda-6 protein. In another embodiment, the expression is measured byin situ hybridization.

This invention also provides a method for indicating the effectivenessof a treatment against cancer comprising measuring the expression levelof mda-6 gene in the cells of the cancer, the increase of the expressionlevel indicating the effectiveness of the treatment. In an embodiment,the cancer is melanoma. In another embodiment, the cancer is leukemia.In a separate embodiment, the cancer is lymphoma. In another embodiment,the cancer is neuroblastoma. In a further embodiment, the cancer is aglioblastoma multiforme tumor. In a still further embodiment, the canceris a carcinoma.

This invention provides an isolated nucleic acid molecule encoding aprotein produced by a melanoma differentiation associated genedesignated mda-7. In an embodiment, the nucleic acid is a cDNA. Inanother embodiment, the nucleic acid is genomic DNA.

In a separate embodiment, the mda-7 gene is coding for a human protein.

This invention provides an isolated nucleic acid molecule of a cDNA,wherein the protein is a human protein.

This invention provides a nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence of thenucleic acid molecule of mda-7.

This invention provides a method of detecting the expression of mda-7gene in a cell comprising: a) isolating the nucleic acids in the cell;b) hybridizing the isolated nucleic acids with the nucleic acidmolecules of at least 15 nucleotides capable of specifically hybridizingwith a sequence of the nucleic acid molecule of an isolated nucleic acidmolecule encoding a protein produced by a melanoma differentiationassociated gene designated mda-7 under conditions permitting hybridsformation; and c) detecting hybrids formed, the detection of the hybridsformed indicating the expression of mda-7 gene in the cell.

This invention provides a method for determining whether a cell is amelanoma cell or a carcinoma cell comprising detecting the expression ofmda-7 gene using the above-described method of detecting the expressionof mda-7 gene in a cell, the expression of mda-7 gene indicating thatthe cell is a melanoma cell.

The invention further provides a method for distinguishing a melanocyteor early stage melanoma cell from an advanced metastatic melanoma cellcomprising detecting the expression of mda-7 gene using theabove-described method, the expression of mda-7 gene indicating that thecell is a melanocyte or early stage melanoma cell.

This invention provides a method for distinguishing a normalneuroectodermal cell from a malignant neuroectodermal cell comprisingdetecting the expression of mda-7 gene using the above-described methodof detecting the expression of mda-7 gene in a cell, the expression ofmda-7 gene indicating that the tissue lineage of the cell is normalneuroectodermal cell.

The invention also provides a method for distinguishing a fibroblastfrom an epithelial cell comprising detecting the expression of mda-7gene using the above-described method, the expression of mda-7 geneindicating that the cell is a fibroblast.

This invention provides a method for identifying a compound capable ofinducing growth suppression in human melanoma cells comprising: a)incubating appropriate concentration of the human melanoma cells with anappropriate concentration of the compound; and b) detecting theexpression of mda-7 using the above-described method, the expression ofmda-7 gene indicating that the compound is capable of inducing growthsuppression in human melanoma cells.

The invention further provides a method for monitoring the response of acell to an anticancer agent such as adriamycin or vincristine comprisingdetecting the expression of mda-7 gene using the above-described method,the expression of mda-7 gene indicating that the cell responds to theanticancer agent.

The invention also provides a method for monitoring the response of acell to DNA damage induced by UV irradiation comprising detecting theexpression of mda-7 gene using the above-described method, theexpression of mda-7 gene indicating that the cell responds to the DNAdamage.

This invention provides a method for reversing the malignant phenotypeof cells comprising: (a) linking the mda-7 gene to a regulatory elementsuch that the expression of the mda-7 gene is under the control of theregulatory element; and (b) introducing the linked mda-7 gene into themalignant cells for the expression of the mda-7 gene, thereby reversingthe malignant phenotype of cells.

This invention also provides a method for reversing the malignantphenotype of cells comprising: (a) linking the mda-7 gene to aregulatory element such that the expression of the mda-7 gene is underthe control of the regulatory element; (b) introducing the linked mda-7gene into the malignant cells; and (c) placing the cells from step (b)in appropriate conditions to express the mda-7 gene such that theexpression of the mda-7 gene will reverse the transforming phenotype ofthe malignant cells.

This invention also provides a method for reversing the phenotype ofmalignant cells in a subject comprising: (a) linking the mda-7 gene to aregulatory element such that the expression of the mda-7 gene is underthe control of the regulatory element; and (b) introducing the linkedmda-7 gene into the malignant cells for the expression of the mda-7gene, thereby reversing the phenotype of the malignant cells.

This invention also provides a method to reversing the phenotype ofmalignant cells in a subject comprising: (a) linking the mda-7 gene to aregulatory element such that the expression of the mda-7 gene is underthe control of the regulatory element; (b) introducing the linked mda-7gene into the malignant cells of the subject; and (c) inducing theexpression of the mda-7 gene which will reverse the transformingproperties of the cells, thereby reversing the phenotype of themalignant cells in the subject.

This invention also provides a method of inducing growth suppression intumorigenic and metastatic cells comprising: (a) linking the mda-7 geneto a regulatory element such that the expression of the mda-7 gene isunder the control of the regulatory element; (b) introducing the linkedmda-7 gene into the tumorigenic and metastatic cells; and (c) inducingthe expression of the mda-7 gene, thereby inducing growth suppression intumorigenic and metastatic cells.

This invention also provides a method of inducing terminaldifferentiation in tumorigenic and metastatic cells comprising: (a)linking the mda-7 gene to a regulatory element such that the expressionof the mda-7 gene is under the control of the regulatory element; (b)introducing the linked mda-7 gene into the tumorigenic and metastaticcells; and (c) inducing the expression of the mda-7 gene, therebyinducing terminal differentiation in tumorigenic and metastatic cells.In an embodiment, the cell is a melanoma cell. In another embodiment,the cell is a leukemia cell. In a further embodiment, the cell is alymphoma cell. In a still further embodiment, the cell is aneuroblastoma cell. In another still further embodiment, the cell is aglioblastoma multiforme cell.

In a separate embodiment, the regulatory element is a promoter. In afurther embodiment, the promoter is a tissue-specific promoter. Inanother embodiment, the promoter is an inducible promoter.

The linked mda-7 gene may be introduced into the cells by naked DNAtechnology, retroviral vectors, antibody-coated liposomes, mechanical orelectrical means. These technologies are known in the art.

This invention also provides a method of determining the stage of amelanoma comprising: (a) obtaining appropriate amount of cells from themelanoma; (b) measuring the expression level of the mda-7 gene in thecells; and (b) comparing the expression level with predeterminedstandards of normal and melanoma cells in different stages, therebydetermining the stage of a melanoma.

In an embodiment, the expression is measured by the antibodies againstthe mda-7 protein. In another embodiment, the expression is measured byin situ hybridization.

This invention also provides a method for indicating the effectivenessof a treatment against cancer comprising measuring the expression levelof mda-7 gene in the cells of the cancer, the increase of the expressionlevel indicating the effectiveness of the treatment. The cancer may be amelanoma, leukemia, lymphoma, neuroblastoma or a glioblastoma multiformetumor.

This invention provides a method of determining whether a cell issenescent comprising detecting the expression of mda-7, the expressionof the mda-7 gene indicating that the cell is senescent.

This invention provides a method of identifying a compound inhibitingsenescence comprising: a) incubating a plurality of cells with anappropriate amount of a compound; b) detecting the expression of mda-7,the inhibition of the expression of mda-7 indicating that the compoundis inhibiting senescence.

This invention provides an isolated nucleic acid molecule encoding aprotein produced by a melanoma differentiation associated genedesignated mda-8. In an embodiment, the nucleic acid is a cDNA. Inanother embodiment, the nucleic acid is genomic DNA.

In a separate embodiment, the mda-8 gene is coding for a human protein.

This invention provides a nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence of thenucleic acid molecule of an isolated nucleic acid molecule encoding aprotein produced by a melanoma differentiation associated genedesignated mda-8.

This invention provides a method of detecting the expression of mda-8gene in a cell comprising: a) isolating the nucleic acids in the cell;b) hybridizing the isolated nucleic acids with the nucleic acidmolecules capable of mda-8, under conditions permitting hybridsformation; and c) detecting hybrids formed, the detection of the hybridsformed indicating the expression of mda-8 gene in the cell.

This invention provides a method for distinguishing a glial cell from amalignant astrocytoma cell comprising detecting the expression of mda-8gene using the above-described method, the expression of mda-8 geneindicating that the cell is a normal glial cell.

The invention also provides a method for monitoring the response of acell to an anticancer agent such as actinomycin D, adriamycin orcis-platinum comprising detecting the expression of mda-8 gene using theabove-described method, the expression of mda-8 gene indicating that thecell responds to the anticancer agent.

The invention further provides a method for monitoring the response of acell to DNA damage induced by UV irradiation comprising detecting theexpression of mda-8 gene using the above-described method, theexpression of mda-8 gene indicating that the cell responds to the DNAdamage.

This invention provides a method for detecting type II interferons in asample comprising: a) incubating the sample with a target cellcontaining the 5' regulatory element of mda-8 permitting binding of typeII interferon transcriptional regulatory proteins to the 5' regulatoryelement; and b) detecting the binding, the binding indicating thepresence of type II interferons in the sample.

In an embodiment, the cell is an eukaryotic cell.

In another embodiment, the 5' regulatory element is linked to the nativemda-8 gene and the detection of binding is by the examination of theelevated level of mda-8 gene expression.

In one embodiment, the 5' regulatory element is linked to a marker gene.In a still further embodiment, the marker gene is β-galactosidase,luciferase or CAT.

This invention provides a method for identifying a compound capable ofinducing terminal differentiation in human melanoma cells comprising: a)inducing appropriate concentration of the human melanoma cells with anappropriate concentration of the compound; and b) detecting theexpression of mda-8 using the above-described method, the expression ofmda-8 gene indicating that the compound is capable of inducing terminaldifferentiation in human melanoma cells.

This invention provides an isolated nucleic acid molecule encoding aprotein produced by a melanoma differentiation associated genedesignated mda-9. In an embodiment, the nucleic acid is a cDNA. Inanother embodiment, the nucleic acid is genomic DNA.

In a separate embodiment, the mda-9 gene is coding for a human protein.

This invention provides an isolated nucleic acid molecule of an isolatednucleic acid molecule encoding a protein produced by a melanomadifferentiation associated gene designated mda-9, wherein the protein isa human protein.

This invention provides a nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence of thenucleic acid molecule of an isolated nucleic acid molecule encoding aprotein produced by a melanoma differentiation associated genedesignated mda-9.

This invention provides a method of detecting the expression of mda-9gene in a cell comprising: a) isolating the nucleic acids in the cell;b) hybridizing the isolated nucleic acids with the nucleic acidmolecules of at least 15 nucleotides capable of specifically hybridizingwith a sequence of the nucleic acid molecule of an isolated nucleic acidmolecule encoding a protein produced by a melanoma differentiationassociated gene designated mda-9 under conditions permitting hybridsformation; and c) detecting hybrids formed, detection of hybrids formedindicating the expression of mda-9 gene in the cell.

This invention provides a method for indicating the stage of progressionof a human melanoma cell comprising detecting the expression of mda-9gene using the method of detecting the expression of mda-9 gene in acell comprising: a) isolating the nucleic acids in the cell; b)hybridizing the isolated nucleic acids with the nucleic acid moleculesof at least 15 nucleotides capable of specifically hybridizing with asequence of the nucleic acid molecule of an isolated nucleic acidmolecule encoding a protein produced by a melanoma differentiationassociated gene designated mda-9 under conditions permitting hybridsformation; and c) detecting hybrids formed, detection of hybrids formedindicating the expression of mda-9 gene in the cell, the expression ofmda-9 gene indicating the stage of progression of the human melanomacell.

This invention provides a method for identifying a compound capable ofinducing terminal differentiation in human melanoma cells comprising: a)incubating appropriate concentration of the human melanoma cells with anappropriate concentration of the compound; b) detecting the expressionof mda-9 using the method of detecting the expression of mda-9 gene in acell comprising: a) isolating the nucleic acids in the cell; b)hybridizing the isolated nucleic acids with the nucleic acid moleculesof at least 15 nucleotides capable of specifically hybridizing with asequence of the nucleic acid molecule of an isolated nucleic acidmolecule encoding a protein produced by a melanoma differentiationassociated gene designated mda-9 under conditions permitting hybridsformation; and c) detecting hybrids formed, detection of hybrids formedindicating the expression of mda-9 gene in the cell, the expression ofmda-9 gene indicating that the compound is capable of inducing terminaldifferentiation in human melanoma cells.

This invention provides a method for identifying a compound capable ofinducing specific patterns of DNA damage caused by UV irradiation andgamma irradiation in human melanoma cells comprising: a) inducingappropriate concentration of the human melanoma cells with anappropriate concentration of the compound; and b) detecting theexpression of mda-9 using the method of detecting the expression ofmda-9 gene in a cell comprising: a) isolating the nucleic acids in thecell; b) hybridizing the isolated nucleic acids with the nucleic acidmolecules of at least 15 nucleotides capable of specifically hybridizingwith a sequence of the nucleic acid molecule of an isolated nucleic acidmolecule encoding a protein produced by a melanoma differentiationassociated gene designated mda-9 under conditions permitting hybridsformation; and c) detecting hybrids formed, detection of hybrids formedindicating the expression of mda-9 gene in the cell, the expression ofmda-9 gene indicating that the compound is capable of inducing specificpatterns of DNA damage caused by UV irradiation and gamma irradiation inhuman melanoma cells.

The invention also provides a method for identifying the presence oftumor necrosis factor or a similarly acting agent comprising detectingthe expression of mda-9 gene using the above-described method, theexpression of mda-9 gene indicating that the tumor necrosis factor orsimilar agent is present.

The invention further provides a method for monitoring the response of acell to an anticancer agent such as phenyl butyrate or VP-16 comprisingdetecting the expression of mda-9 gene using the above-described method,the expression of mda-9 gene indicating that the cell responds to theanticancer agent.

In a separate embodiment, the mda-9 gene is coding for a human protein.

This invention provides a nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence of thenucleic acid molecule of mda-9.

This invention provides a method of detecting the expression of mda-9gene in a cell comprising: a) isolating the nucleic acids in the cell;b) hybridizing the isolated nucleic acid with the nucleic acid capableof specifically hybridizing with mda-9 under conditions permittinghybrid formation and c) detecting hybrids formed, detection of hybridsformed indicating the expression of mda-9 gene in the cell.

This invention provides a method for distinguishing an early stage andmore progressed human melanoma cell comprising detecting the expressionof mda-9 gene indicating that the cell is more progressed human melanomacell.

This invention provides a method for identifying a compound capable ofinducing terminal differentiation in human melanoma cells comprising: a)incubating human melanoma cell with the compound effective of inducingterminal differentiation in human melanoma cells; and b) detecting theexpression of mda-9 using the above-described method, the expression ofmda-9 gene indicating that the compound is capable of inducing terminaldifferentiation in human melanoma cells.

This invention provides a method for identifying a compound capable ofinducing specific patterns of DNA damage caused by UV irradiation andgamma irradiation in human melanoma cells comprising: a) incubatinghuman melanoma cells with the compound effective of inducing specificpatterns of DNA damage caused by UV irradiation and gamma irradiation inhuman melanoma cells; and b) detecting the expression of mda-90 usingthe above-described method, the expression of mda-9 gene indicating thatthe compound is capable of inducing specific patterns of DNA damagecaused by UV irradiation and gamma irradiation in human melanoma cells.

This invention provides an isolated nucleic acid molecule encoding aprotein produced by a melanoma differentiation associated genedesignated mda-11. In an embodiment, the nucleic acid is a cDNA. Inanother embodiment, the nucleic acid is genomic DNA.

In a separate embodiment, the mda-11 gene is coding for a human protein.

This invention provides an isolated nucleic acid molecule encoding aprotein produced by a melanoma differentiation associated genedesignated mda-14. In an embodiment, the nucleic acid is a cDNA. Inanother embodiment, the nucleic acid is genomic DNA.

In a separate embodiment, the mda-14 gene is coding for a human protein.

This invention provides an isolated nucleic acid molecule encoding aprotein produced by a melanoma differentiation associated genedesignated mda-17. In an embodiment, the nucleic acid is a cDNA. Inanother embodiment, the nucleic acid is genomic DNA.

In a separate embodiment, the mda-17 gene is coding for a human protein.

This invention provides an isolated nucleic acid molecule encoding aprotein produced by a melanoma differentiation associated genedesignated mda-18. In an embodiment, the nucleic acid is a cDNA. Inanother embodiment, the nucleic acid is genomic DNA.

In a separate embodiment, the mda-18 gene is coding for a human protein.

In an embodiment, the above-described isolated nucleic acid molecule iscDNA. In another embodiment, the nucleic acid molecule is from humans.

This invention also encompasses DNAs and cDNAs which encode amino acidsequences which differ from those of protein encoded by the melanomadifferentiation associated genes, but which should not producephenotypic changes. Alternatively, this invention also encompasses DNAsand cDNAs which hybridize to the DNA and cDNA of the subject invention.Hybridization methods are well known to those of skill in the art.

The DNA molecules of the subject invention also include DNA moleculescoding for polypeptide analogs, fragments or derivatives of antigenicpolypeptides which differ from naturally-occurring forms in terms of theidentity or location of one or more amino acid residues (deletionanalogs containing less than all of the residues specified for theprotein, substitution analogs wherein one or more residues specified arereplaced by other residues and addition analogs where in one or moreamino acid residues is added to a terminal or medial portion of thepolypeptides) and which share some or all properties ofnaturally-occurring forms. These molecules include: the incorporation ofcodons "preferred" for expression by selected non-mammalian hosts; theprovision of sites for cleavage by restriction endonuclease enzymes; andthe provision of additional initial, terminal or intermediate DNAsequences that facilitate construction of readily expressed vectors.

The DNA molecules described and claimed herein are useful for theinformation which they provide concerning the amino acid sequence of thepolypeptide and as products for the large scale synthesis of thepolypeptide by a variety of recombinant techniques. The molecule isuseful for generating new cloning and expression vectors, transformingand transfecting prokaryotic and eukaryotic host cells, and new anduseful methods for cultured growth of such host cells capable ofexpression of the polypeptide and related products.

Moreover, the isolated nucleic acid molecules encoding a protein codedby the melanoma differentiation associated gene are useful for thedevelopment of probes to study melanoma differentiation. In addition,the isolated nucleic acid molecules encoding a protein coded by themelanoma differentiation associated gene are useful for screening foragents with anticancer activity, agents which can induce DNA damage andagents which induce cell growth arrest in human melanoma.

The isolated nucleic acid molecules encoding a protein coded by themelanoma differentiation associated gene are also useful fordistinguishing normal from malignant central nervous system cells,adenocarcinomas from carcinomas, and fibroblasts from epithelial cells.

This invention further provides a nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence of theabove-described nucleic acid molecule, i.e., the melanomadifferentiation associated genes.

This nucleic acid molecule produced can either be DNA or RNA. As usedherein, the phrase "specifically hybridizing" means the ability of anucleic acid molecule to recognize a nucleic acid sequence complementaryto its own and to form double-helical segments through hydrogen bondingbetween complementary base pairs.

When a situation arises that requires the nucleic acid molecule to beuniquely recognizing a gene, it is well-known in the art to selectregions in the sequence which will distinguish one gene from the other.Simple experiments may be designed to find such unique regions.

This nucleic acid molecule of at least 15 nucleotides capable ofspecifically hybridizing with a sequence of the above-described nucleicacid molecule can be used as a probe. Nucleic acid probe technology iswell known to those skilled in the art who will readily appreciate thatsuch probes may vary greatly in length and may be labeled with adetectable label, such as a radioisotope or fluorescent dye, tofacilitate detection of the probe. DNA probe molecules may be producedby insertion of a DNA molecule which encodes a protein produced by amelanoma differentiation associated gene into suitable vectors, such asplasmids or bacteriophages, followed by transforming into suitablebacterial host cells, replication in the transformed bacterial hostcells and harvesting of the DNA probes, using methods well known in theart. Alternatively, probes may be generated chemically from DNAsynthesizers.

RNA probes may be generated by inserting the above-described isolatednucleic acid molecule downstream of a bacteriophage promoter such as T3,T7 or SP6. Large amounts of RNA probe may be produced by incubating thelabeled nucleotides with the linearized fragment containing theabove-described molecule where it contains an upstream promoter in thepresence of the appropriate RNA polymerase.

This invention also provides a method of detecting expression of amelanoma differentiation associated gene in a cell which comprisesobtaining total cellular RNA or mRNA from the cell, contacting the totalcellular RNA or mRNA so obtained with a labelled nucleic acid moleculeof at least 15 nucleotides capable of specifically hybridizing with asequence of the above-described nucleic acid molecule under hybridizingconditions, determining the presence of total cellular RNA or mRNAhybridized to the molecule, and thereby detecting the expression of themelanoma differentiation associated gene in the cell.

The nucleic acid molecules synthesized above may be used to detectexpression of melanoma differentiation associated genes by detecting thepresence of the correspondent RNA or mRNA. Total cellular RNA or mRNAfrom the cell may be isolated by many procedures well known to a personof ordinary skill in the art. The hybridizing conditions of the labellednucleic acid molecules may be determined by routine experimentation wellknown in the art. The presence of total cellular RNA or mRNA hybridizedto the probe may be determined by gel electrophoresis or other methodsknown in the art. By measuring the amount of the hybrid made, theexpression of the melanoma differentiation associated genes by the cellcan be determined. The labelling may be radioactive. For an example, oneor more radioactive nucleotides can be incorporated in the nucleic acidwhen it is made.

In one embodiment of this invention, nucleic acids are extracted fromlysed cells and the mRNA is isolated from the extract using an oligo-dTcolumn which binds the poly-A tails of the mRNA molecules. The mRNA isthen exposed to radioactively labelled probe on a nitrocellulosemembrane, and the probe hybridizes to and thereby labels complementarymRNA sequences. Binding may be detected by luminescence autoradiographyor scintillation counting. However, other methods for performing thesesteps are well known to those skilled in the art, and the discussionabove is merely an example.

This invention also provides a method of detecting expression of amelanoma differentiation associated gene in tissue sections whichcomprises contacting the tissue sections with a labelled nucleic acidmolecule of at least 15 nucleotides capable of specifically hybridizingwith a sequence of the above-described nucleic acid molecule underhybridizing conditions, determining the presence of mRNA hybridized tothe molecule, and thereby detecting the expression of the melanomadifferentiation associated gene in tissue sections.

This invention also provides the above-describe nucleic acid moleculesoperatively linked to a promoter of RNA transcription.

This invention provides vectors which comprise the above-describedisolated nucleic acid molecules. In an embodiment, the vector is aplasmid.

Various vectors including plasmid vectors, cosmid vectors, bacteriophagevectors and other viruses are well known to ordinary skilledpractitioners. This invention further provides a vector which comprisesthe isolated nucleic acid molecule encoding a protein produced by amelanoma differentiation gene.

As an example to obtain these vectors, insert and vector DNA can both beexposed to a restriction enzyme to create complementary ends on bothmolecules which base pair with each other and are then ligated togetherwith DNA ligase. Alternatively, linkers can be ligated to the insert DNAwhich correspond to a restriction site in the vector DNA, which is thendigested with the restriction enzyme which cuts at that site. Othermeans are also available and known to an ordinary skilled practitioner.

In an embodiment, the nucleic acid molecule is cloned in the XhoI/EcoRIsite of pBlueScript. Plasmids, mda-1, mda-4, mda-5, mda-6, mda-7, mda-8,mda-9, mda-11, mda-14, mda-17, and mda-18 were deposited on Oct. 26,1993 with the American Type Culture Collection (ATCC), 12301 ParklawnDrive, Rockville, Md. 20852, U.S.A. under the provisions of the BudapestTreaty for the International Recognition of the Deposit of Microorganismfor the Purposes of Patent Procedure. Plasmids, mda-1, mda-4, mda-5,mda-6, mda-7, mda-8, mda-9, mda-11, mda-14, mda-17, and mda-18 wereaccorded ATCC Accession Numbers 75582, 75583, 75584, 75585, 75586,75587, 75588, 75589, 75590, 75591 and 75592 respectively.

In another embodiment, a 3' fragment of the mda-6 gene is cloned in theEcoRI and XbaI site of the pBluescript plasmid and designed as mda-6.3'.The mda-6.3' was deposited on Sep. 30, 1994 with the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852,U.S.A. under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganism for the Purposes of PatentProcedure. Plasmid, mda-6.3' was accorded ATCC Accession Numbers 75903.

In another embodiment, a 5' fragment of the mda-6 gene is cloned in thesalI site of the pSP64 plasmid and designed as mda-6.5'. The mda-6.5'was deposited on Sep. 30, 1994 with the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. under theprovisions of the Budapest Treaty for the International Recognition ofthe Deposit of Microorganism for the Purposes of Patent Procedure.Plasmid, mda-6.5' was accorded ATCC Accession Number 75904.

Plasmids mda-6.3' and mda-6.5' constitute the full-length of the mda-6gene. An ordinary skilled artisan can easily obtain the inserts from theplasmids and ligate the inserts to obtain the full-length gene.

In another embodiment, a 3' fragment of the mda-7 gene is cloned in theEcoRI and XbaI site of the pBluescript plasmid and designed as mda-7.3'.The mda-7.3' was deposited on Sep. 30, 1994 with the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852,U.S.A. under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganism for the Purposes of PatentProcedure. Plasmid, mda-7.3' was accorded ATCC Accession Number 75905.

In another embodiment, a 5' fragment of the mda-7 gene is cloned in thesalI site of the pSP64 plasmid and designed as mda-7.5'. The mda-7.5'was deposited on Sep. 30, 1994 with the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. under theprovisions of the Budapest Treaty for the International Recognition ofthe Deposit of Microorganism for the Purposes of Patent Procedure.Plasmid, mda-7.5' was accorded ATCC Accession Numbers 75906.

Plasmids mda-7.3' and mda-7.5' constitute the full-length of the mda-7gene. An ordinary skilled artisan can easily obtain the inserts from theplasmids and ligate the inserts to obtain the full-length gene.

This invention provides a host vector system for the production of apolypeptide having the biological activity of a protein encoded bymelanoma differentiation associated gene which comprises theabove-described vector and a suitable host. These vectors may betransformed into a suitable host cell to form a host cell vector systemfor the production of a protein produced by the melanoma differentiationassociated genes.

Regulatory elements required for expression include promoter sequencesto bind RNA polymerase and transcription initiation sequences forribosome binding. For example, a bacterial expression vector includes apromoter such as the lac promoter and for transcription initiation theShine-Dalgarno sequence and the start codon AUG. Similarly, a eukaryoticexpression vector includes a heterologous or homologous promoter for RNApolymerase II, a downstream polyadenylation signal, the start codon AUG,and a termination codon for detachment of the ribosome. Such vectors maybe obtained commercially or assembled from the sequences described bymethods well known in the art, for example the methods described abovefor constructing vectors in general. Expression vectors are useful toproduce cells that express the protein produced by the melanomadifferentiation associated genes.

This invention further provides an isolated DNA or cDNA moleculedescribed hereinabove wherein the host cell is selected from the groupconsisting of bacterial cells (such as E.coli), yeast cells, fungalcells, insect cells and animal cells. Suitable animal cells include, butare not limited to Vero cells, HeLa cells, Cos cells, CV1 cells andvarious primary mammalian cells.

As stated above and in the text which follows, this invention providesgene(s) which express when a cell becomes terminally differentiated andirreversibly growth arrested, treated with a DNA damaging agent and/orexposed to an anticancer agent. Therefore, this invention is useful forinducing a target cell to a terminally differentiated stage. Such atarget cell may be a cancerous cell such as a melanoma cell or aglioblastoma multiforme cell. The gene which expresses when a cellbecomes terminally differentiated may be introduced into the target cellvia retroviral technology or other technologies known in the art. Thegene may be controlled by its own promoter or other heterologouspromoters. Expression of this gene will then result in terminaldifferentiation and an irreversible loss of proliferative capacity inthe cancerous cell.

This invention has also provided nucleic acid molecules which willsuppress the terminal differentiation of a cell. Such molecules areuseful for preventing terminal differentiation by switching off a targetgene in a cell which has been treated with differentiation inducingagents, anticancer agents or DNA damaging agents. The target gene may beswitched off via antisense technology. After the gene has been switchedoff, normal cells, such as bone marrow stem cells, can be prevented frombecoming terminally differentiated and irreversibly growth arrested whentreated with differentiation inducing agents, anticancer agents or DNAdamaging agents.

Antisense technology is well known in the art. Essentially, a segment ofthe melanoma differentiation associated gene will be selected to be theantisense sequence. The expression of the antisense sequence will switchoff the expression of the gene. The antisense sequence may be introducedinto the cell via technologies which are well known in the art, such aselectroporation transduction, retroviral insertion or liposome-mediatedgene transfer.

This invention also provides a method of producing a polypeptide havingthe biological activity of a protein encoded by melanoma differentiationassociated gene which comprises growing the host cells of theabove-described host vector system under suitable conditions permittingproduction of the polypeptide and recovering the polypeptide soproduced.

This invention provides a mammalian cell comprising a DNA moleculeencoding a protein produced by a melanoma differentiation associatedgene, such as a mammalian cell comprising a plasmid adapted forexpression in a mammalian cell, which comprises a DNA molecule encodinga protein produced by the melanoma differentiation associated gene andthe regulatory elements necessary for expression of the DNA in themammalian cell so located relative to the DNA encoding a proteinproduced by a melanoma differentiation gene as to permit expressionthereof.

Numerous mammalian cells may be used as hosts, including, but notlimited to, the mouse fibroblast cell NIH3T3, CREF cells, CHO cells,HeLa cells, Ltk⁻ cells, Cos cells, etc. Expression plasmids such as thatdescribed supra may be used to transfect mammalian cells by methods wellknown in the art such as calcium phosphate precipitation,electroporation or the plasmid may be otherwise introduced intomammalian cells, e.g., by microinjection, to obtain mammalian cellswhich comprise DNA, e.g., cDNA or a plasmid, encoding a protein producedby a melanoma differentiation associated gene.

Also provided by this invention is a purified protein encoded by theabove-described isolated nucleic acid molecule. As used herein, the term"purified protein" shall mean isolated naturally-occurring proteinencoded by a melanoma differentiation associated gene (purified fromnature or manufactured such that the primary, secondary and tertiaryconformation, and posttranslational modifications are identical tonaturally-occurring material) as well as non-naturally occurringpolypeptides having a primary structural conformation (i.e. continuoussequence of amino acid residues). Such polypeptides include derivativesand analogs.

This invention also provides a method to produce antibody using theabove purified protein. In an embodiment, the antibody is monoclonal. Inanother embodiment, the antibody is polyclonal.

This invention further provides antibodies capable of binding to thepurified protein produced by the melanoma differentiation associatedgenes.

With the protein sequence information which can either be derived fromthe above described nucleic molecule or by direct protein sequencing ofthe above described purified protein, antigenic areas may be identifiedand antibodies directed against these areas may be generated andtargeted to the cancer for imaging the cancer or therapies.

This invention provides a method to select specific regions on theprotein produced by a melanoma differentiation associated gene togenerate antibodies. Amino acid sequences may be analyzed by methodswell known to those skilled in the art to determine whether they producehydrophobic or hydrophilic regions in the proteins which they build. Inthe case of cell membrane proteins, hydrophobic regions are well knownto form the part of the protein that is inserted into the lipid bilayerof the cell membrane, while hydrophilic regions are located on the cellsurface, in an aqueous environment. Usually, the hydrophilic regionswill be more immunogenic than the hydrophobic regions. Therefore thehydrophilic amino acid sequences may be selected and used to generateantibodies specific to protein produced by the melanoma differentiationgenes. The selected peptides may be prepared using commerciallyavailable machines. As an alternative, DNA, such as a cDNA or a fragmentthereof, may be cloned and expressed and the resulting polypeptiderecovered and used as an immunogen.

Polyclonal antibodies against these peptides may be produced byimmunizing animals using the selected peptides. Monoclonal antibodiesare prepared using hybridoma technology by fusing antibody producing Bcells from immunized animals with myeloma cells and selecting theresulting hybridoma cell line producing the desired antibody.Alternatively, monoclonal antibodies may be produced by in vitrotechniques known to a person of ordinary skill in the art. Theseantibodies are useful to detect the expression of protein produced bythe melanoma differentiation associated genes in living animals, inhumans, or in biological tissues or fluids isolated from animals orhumans.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

EXPERIMENTAL DETAILS First Series of Experiments Materials and Methods

Cell Line, Growth Conditions, and Preparation of Conditioned Medium

The HO-1 cell line is a melanotic melanoma derived from a 49-year-oldfemale and was used between passage 100 and 125 (6,8,13). HO-1 cellswere kindly provided by Dr. Beppino C. Giovanella, Stehlin Foundationfor Cancer Research, Houston, Tex. Cultures were grown at 37° C. in a95% air 5% CO₂ -humidified incubator in Dulbecco's modified Eagle'smedium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10)(Hyclone, Logan, Utah). HO-1 cells were maintained in the logarithmicstage of growth by subculturing (1:5 or 1:10) prior to confluencyapproximately every 4 to 5 days. The effect of IFN-β (2000 units/ml),IFN-γ (2000 units/ml), IFN-β+IFN-γ (1000 units/ml of each interferon),MPA (3.0 μM), RA (2.5 μM), MEZ (10 ng/ml), IFN-β+MEZ (2000 units/ml+10ng/ml), MPA+MEZ (3.0 μM+10 ng/ml, and RA+MEZ (2.5 μM+10 ng/ml) on growthwas determined after 4 days of treatment, as described previously(6,8,9). Terminal cell differentiation, with a concomitant loss ofproliferative capacity, was assayed by treating cells with the variousagents for either 4 or 7 days, washing cultures two times withserum-free DMEM, followed by incubation in DMEM-10 in the absence ofinducer(s) for an additional 14 days. Total cell numbers were determinedafter days 4, 7, 14, and 21, using a Z_(M) Coulter Counter, and viablecell numbers were determined by trypan blue dye exclusion (6). Terminalcell differentiation was indicated if no proliferation occurred, butcells remained viable, after growth for 14 days, with medium changesevery 3 or 4 days, in the absence of the inducer(s). Increases in cellnumber (two or more population doublings) after removal of the inducingagent(s) was considered reversible growth suppression. Conditionedmedium was prepared from HO-1 cells treated with a high dose of MEZ (50ng/ml), IFN-β+MEZ (2000 U/ml+10 ng/ml), MPA+MEZ (3.0 μM+10 ng/ml), orRA+MEZ (2.5μ+10 ng/ml) for 24 hours followed by three washes in DMEMwithout FBS and growth for 72 hours in inducer-free medium (DMEM-10).Conditioned medium was collected from treated cultures, contaminatingcells were removed by centrifugation at 1000 rpm/10 min., andconditioned medium was stored at 4° C. until assayed for gene modulatoryactivity. Control conditioned medium was obtained as experimentalconditioned medium from cells receiving only a medium change 24 hoursafter plating in the absence of test compound(s) and growth for 72 hoursin DMEM-10.

RNA Isolation and Northern Hybridization Analysis

Steady-state levels of specific mRNAs were determined by Northernblotting analysis of total RNA probed with appropriate ³² P-labeled geneprobes as described previously (14-16). RNAs were analyzed from cellstreated with inducer for 24 hours, treated for 24 hours with inducerfollowed by growth for 72 hours in the absence of inducer, or treatedcontinuously for 96 hours with the inducer. The concentration ofinducing compounds used were the same as those used for growth studies.The effect of conditioned medium on gene expression changes wasdetermined by treating HO-1 cells for 24 hours with a 1:2 dilution ofconditioned medium (equal volumes of conditioned medium and DMEMsupplemented with 10% fetal bovine serum (DMEM-10) or for 96 hours witha 1:4 dilution of conditioned medium (1 vol. of conditioned medium to 3vol. of DMEM-10). The probes used in the present study were specific forβ-actin (17), γ-actin (17), c-jun (18), c-myc (19), fibronectin (20),gro/MGSA (21), HLA Class I antigen (22), HLA Class II antigen(HLA-DR.sub.β) (22), α₅ integrin (23), β₁ integrin (24), ISG-15 (25),ISG-54 (25), jun-B (26), and tenascin (27). Northern blots were alsoprobed with a ³² P-labeled GAPDH gene (15) to verify similar mRNAexpression under the various experimental conditions. Followinghybridization, the filters were washed and exposed for autoradiography.

Reagents

Recombinant human IFN-β, with a serine substituted for a cysteine atposition 17 of the molecule (28), was kindly provided by TritonBioscience (Alameda, Calif.). IFN-β was obtained as a lyophilized powderwith a concentration of 4.5×10⁷ U/ml. Recombinant human immuneinterferon (IFN-γ) was produced and purified to homogeneity as describedpreviously (29). IFN-γ was kindly provided by Dr. Sidney Pestka,UMDNJ-Robert Wood Johnson Medical School, Piscataway, N.J. Theinterferon titers were determined using a cytopathic effect inhibitionassay with vesicular stomatitis virus on a bovine kidney cell line(MDBK) or human fibroblast AG-1732 cells (30). Concentrated stocks ofIFN-β and IFN-γ were diluted to 1×10⁶ U/ml in DMEM-10, frozen at -80°C., thawed immediately prior to use, and diluted to the appropriateconcentration in DMEM-10. MEZ, RA, and MPA were obtained from SigmaScientific Co. (St. Louis, Mo.). Stock solutions were prepared indimethyl sulfoxide (DMSO), aliquoted into small portions, and stored at-20° C. The final concentration of DMSO used in solvent controls was0.01%. This concentration of DMSO did not alter growth, melaninsynthesis, tyrosinase activity, or antigen expression in HO-1 cells.

Experimental Results

Induction of Reversible and Irreversible Growth Suppression (TerminalCell Differentiation) in HO-1 Cells

The effect of RA, MPA, MEZ, IFN-β and IFN-γ, used alone or in variouscombinations, on growth (reversible and irreversible growthsuppression), melanin synthesis, tyrosinase activity, and cellularmorphology of HO-1 cells is summarized in Table 1. The most effectiveagents in inhibiting HO-1 growth were the combinations of IFN-β+MEZ andIFN-β+IFN-γ (FIG. 1). The relative order of antiproliferative activityof the remaining compounds was MPA=MPA+MEZ>IFN-γ>MEZ=RA+MEZ. In the caseof RA, no inhibition of growth occurred. Treatment of HO-1 cells withIFN-β+MEZ for 96 hours resulted in an irreversible loss of proliferativecapacity, that is, terminal cell differentiation. This was indicated bythe failure of treated cells to resume growth, even though they remainedviable, after removal of the test agents. In contrast, all of the othercompounds resulted in a reversible inhibition of growth (data notshown). These findings indicate a dissociation between growthsuppression and terminal cell differentiation in HO-1 cells.

Treatment of HO-1 cells with MPA, MEZ, RA+MEZ, MPA+MEZ, or IFN-β+MEZresulted in distinctive morphologic changes that were characterized bydendrite-like processes (data not shown) (6,8,10). RA, MPA, MEZ, IFN-βand IFN-β+MEZ have been shown previously to enhance melanin synthesis, amarker of melanoma differentiation, in HO-1 cells (6,8,12). In contrast,IFN-γ, alone or in combination with IFN-β, did not induce morphologicchanges or increase melanin levels above that induced by IFN-β alone(10,12). With the exception of IFNβ+MEZ, morphologic and melanin changeswere reversible following removal of the test agent(s) (data not shown).These data indicate that specific cellular and biochemical changesinduced in HO-1 cells, such as reversible growth suppression, melaninsynthesis, and morphologic changes, can occur with or without theinduction of terminal cell differentiation. However, an irreversibleloss of proliferative capacity with the retention of cell viability is aproperty unique to the terminal cell differentiation phenotype.

                  TABLE 1                                                         ______________________________________                                        Experimental                                                                             Morphology   Melanin  Tyrosinase                                     Conditions.sup.a changes.sup.b Synthesis.sup.c activity.sup.d               ______________________________________                                          RA (2.5 μM) - 1+ 2+                                                        MPA (3.0 μM) + 2+ 3+                                                       MEZ + 1+ NT                                                                   (10 ng/ml)                                                                    IFN-β - 1+ NT                                                            (2000 U/ml)                                                                   IFN-γ - - NT                                                            (2000 U/ml)                                                                   RA + MEZ + NT NT                                                              (2.5 μM +                                                                  10 ng/ml)                                                                     MPA + MEZ + NT NT                                                             (3.0 μM +                                                                  10 ng/ml)                                                                     IFN-β + IFN-γ - 1+ NT                                              (1000 U/ml +                                                                  1000 U/ml)                                                                    IFN-β + MEZ  + 4+ NT                                                     (2000 U/ml +                                                                  10 ng/ml)                                                                   ______________________________________                                                      Growth                                                            Experimental Suppression Terminal cell                                        conditions.sup.a (reversible).sup.e differentiation.sup.f                   ______________________________________                                          RA (2.5 μM) - -                                                            MPA (3.0 μM) 3+ -                                                          MEZ 1+ -                                                                      (10 ng/ml)                                                                    IFN-β 3+ -                                                               (2000 U/ml)                                                                   IFN-γ 2+ -                                                              (2000 U/ml)                                                                   RA + MEZ 1+ -                                                                 (2.5 μM +                                                                  10 ng/ml)                                                                     MPA + MEZ 3+ -                                                                (3.0 μM +                                                                  10 ng/ml)                                                                     IFN-β + IFN-γ                                                      (1000 U/ml +                                                                  1000 U/ml)                                                                    IFN-β + MEZ  4+.sup.g +                                                  (2000 U/ml +                                                                  10 ng/ml)                                                                   ______________________________________                                         .sup.a HO1 cells were grown for 96 hr or for 6 or 7 days (with medium         changes after 3 or 4 days) in the presence of the agents indicated. For       morphology, cells grown for 96 hr in the test agent were observed             microscopically. For melanin synthesis, results are for 6d assays for RA      and MPA (11) or 7d assays for MEZ, IFNβ, IFNγ, IFNβ +         IFNγ and IFNβ + MEZ (6,12). For tyrosinase assays, results are     for 6d assays for RA, MPA and MEZ (10). Growth  # suppression (reversible     and terminal cell differentiation) assays, refer to cultures treated with     the indicated compound(s) for 96 hr prior to cell number determination, o     treated for 96 hr and then grown for 2 weeks (with medium changes every 4     days) in the absence of compound prior to cell number determination.          .sup.b Morphology changes refer to the development of dendritelike            processes 96 hr after growth in the indicated compound. + = presence of       dendritelike processes; - = no dendritelike processes.                        .sup.c Melanin assays were determined as described in refs. 6,8,11,12.        Results are expressed as relative increases based on separate data            presented in refs. 6,8,11,12. N.T. = not tested.                              .sup.d Tyrosinase assays were performed as described in ref. 11. Relative     increases (of a similar magnitude) were found for RA, MPA and MEZ after 6     days exposure to these agents (11). N.T. = not tested.                        .sup.e Reversible growth suppression indicates resumption of cell growth      after treatment with the indicated compound(s) for 96 hr, removal of the      test agent and growth for 14 days in compound(s) free medium. Further         details can be found in materials and methods. The degree of initial 96 h     growth suppression is indicated as: - = no significant change in growth       (<10% reduction in growth in comparison with untreated control cultures);     1+  # ˜30% reduction in growth in comparison with untreated control     cultures; 2+ = ˜40% reduction in growth in comparison with untreate     control cultures; 3+ = ˜50 to 60% reduction in growth in comparison     with untreated control cultures; 4+ = ˜80% reduction in growth in       comparison with untreated control cultures.                                   .sup.f The combination of IFNβ + MEZ results in irreversible growth      suppression.                                                                  .sup.g The combination of IFNβ + MEZ results in irreversible growth      suppression                                                              

Changes in the Expression of Early Growth Response andInterferon-responsive Genes During Reversible and Irreversible GrowthSuppression (Terminal Cell Differentiation) in HO-1 Cells

Initial studies were conducted to determine the effect of the variousdifferentiation and growth modulating agents on the 96-hour expressionof the early response genes c-fos, c-jun, jun-B, jun-D, and c-myc (FIG.2). None of the experimental treatments resulted in altered c-fosexpression and no hybridization was obtained with RNA isolated fromcontrol or treated cells probed with jun-D (data not shown). Increaseswere observed, however, in both c-jun and jun-B expression in HO-1 cellstreated for 96 hours with all of the test agents, with the exception ofIFN-β and RA (see FIG. 2). The magnitude of the increase was similar inHO-1 cells treated with IFN-γ, MEZ, or MPA and was greatest for HO-1cells treated with IFN-β+MEZ, MPA+MEZ, or RA+MEZ (see FIG. 2). Unlikec-jun and jun-B expression c-myc expression was downregulated in HO-1cells grown for 96 hours in MEZ, MPA, IFN-β+MEZ, MPA+MEZ, and RA+MEZ(FIG. 2). The magnitude of suppression was greater in HO-1 cells treatedwith IFN-β+MEZ, MPA+MEZ, and RA+MEZ. In contrast, treatment of HO-1cells with IFN-γ, alone or in combination with IFN-β, resulted inincreased c-myc expression.

To determine the temporal relationship between treatment with IFN-β+MEZ,MPA+MEZ, and RA+MEZ and changes in c-jun, jun-B, and c-myc levels, HO-1cells were treated with the inducers for 24 h and total cytoplasmic RNAwas isolated and analyzed by Northern blotting (FIG. 3). Because many ofthe effects observed when MEZ is combined with IFN-β, MPA, or RA areobserved to a lesser extent in cells treated with 10 mg/ml of MEZ alone,RNA was also isolated from HO-1 cell treated for 24 h with a high doseof MEZ (50 ng/ml) (FIG. 3). Under these experimental conditions, c-junand jun-B expression were induced under all experimental conditions andc-myc expression was only marginally reduced in cultures treated withIFN-β+MEZ, MPA+MEZ, and RA+MEZ. Twenty-four-hour exposure to IFN-β+MEZresulted in the largest induction in c-jun and jun-B expression.

To determine if the gene expression changes induced during the inductionof reversible and irreversible growth suppression/differentiationpersist in HO-1 cells treated with specific inducers, cultures weregrown for 24 h in the presence of inducer and then incubated for anadditional 72 h in inducer-free medium prior to isolating total cellularRNA (FIG. 3). Under these experimental conditions, c-jun and jun-Bexpression were induced to the greatest extent in IFN-β+MEZ-treatedcultures. Smaller increases in c-jun and jun-B expression were apparentin high-dose MEZ-, MPA+MEZ-, and RA+MEZ-treated HO-1 cells. In the caseof c-myc, expression was dramatically reduced in IFN-β+MEZ-treatedcultures and reduced to a lesser extent in MPA+MEZ- and RA+MEZ-treatedcultures. These results suggest that the hierarchy for inducing c-jun,jun-B, and c-myc gene expression changes in HO-1 cells isIFN-β+MEZ>MPA+MEZ>RA+MEZ. As will be discussed, this same pattern ofpotency in inducing gene expression changes in HO-1 cells is alsoobserved with a number of additional genes.

Treatment of HO-1 cells for 96 h with the combination of IFN-β+MEZ,MPA+MEZ, or RA+MEZ resulted in the enhanced expression of thecytokine-responsive genes HLA Class I antigen and gro/MGSA (FIG. 2). Incontrast, treatment with IFN-β, MEZ, MPA, or RA alone did notsignificantly alter HLA Class I antigen gene expression or inducegro/MGSA gene expression. Treatment of HO-1 cells for 96 h with IFN-γ,alone or in combination with IFN-β, also enhanced HLA Class I antigenexpression in HO-1 cells, whereas it did not induce gro/MGSA expression(FIG. 2). In contrast, although a 96-h exposure of HO-1 cells to IFN-γ,alone and in combination with IFN-β, enhanced expression of the HLAClass II antigen gene (HLA-DR.sub.β), expression of this gene was notsignificantly enhanced by treatment with IFNβ+MEZ, MPA+MEZ, or RA+MEZ(FIG. 2). These observations indicate possible autocrine loops involvingboth a type I interferon (leukocyte interferon IFN-α) and IFN-β asopposed to a type II interferon (IFN-γ) and gro/MGSA in the induction ofgene expression changes occurring during both reversible (MPA+MEZ andRA+MEZ) and terminal cell differentiation (IFN-β+MEZ).

To determine if an interferon or an interferon-like molecule might beassociated with the induction of reversible or irreversibledifferentiation or both in HO-1 cells, the effect of the variousdifferentiation-inducing and growth-suppressing agents on expression ofthe interferon-responsive genes, ISG-15 and ISG-54 (25, 31, 32) weredetermined. As can be seen in FIG. 2, treatment of HO-1 cells for 96 hwith the combination of IFN-β+MEZ, MPA+MEZ, or RA+MEZ resulted in theinduction in ISG-15 and ISG-54 gene expression.

Further support for a type I interferon and a gro/MGSA autocrine loop inthe reversible commitment to differentiation (MPA+MEZ) and terminal celldifferentiation (IFN-β+MEZ) processes in HO-1 cells was indicated byanalysis of gene expression changes occurring in cultures treated withinducers for 24 h or in cultures treated with inducers for 24 h followedby growth in the absence of inducers for 72 h (FIG. 3). Growth of HO-1cells for 24 h in the presence of IFN-β+MEZ resulted in the induction orenhanced expression of the gro/MGSA, HLA Class I antigen, and ISG-15gene. Similarly, a 24-h treatment with MPA+MEZ induced expression ofgro/MGSA to a similar extent as IFN-β+MEZ, whereas the effects on HLAClass I antigen and ISG-15 expression were more modest. In contrast, a24-h treatment with RA+MEZ or a high dose of MEZ did not induce gro/MGSAor ISG-15 expression, but these treatments did induce a modest increasein HLA Class I antigen expression (FIG. 3). Treatment of HO-1 cells for24 h with inducer (IFN-β+MEZ, MPA+MEZ, RA+MEZ or a high dose of MEZ)followed by growth for 72 hours in the absence of inducer resulted inthe following changes in HO-1 gene expression: (1) gro/MGSA was inducedonly by IFN-β+MEZ treatment; (2) enhanced HLA Class I antigen expressionwas induced by all of the treatments with the following potencies,IFN-β+MEZ>MPA+MEZ ≦ high dose MEZ>RA+MEZ; and (3) ISG-15 was induced toa similar extent by IFN-β+MEZ and MPA+MEZ, whereas RA+MEZ and high-doseMEZ did not induce ISG-15 expression.

Changes in the Expression of Extracellular and Extracellular MatrixReceptor Genes During Reversible and Irreversible Growth Suppression(Terminal Cell Differentiation) in HO-1 cell.

Terminal differentiation in HO-1 cells induced by IFN-β+MEZ isassociated with morphologic changes resulting in the formation ofdendrite-like processes and specific biochemical changes, that is,enhanced tyrosinase activity and melanin synthesis (6). Similarmorphologic and biochemical changes are induced in HO-1 cells by MEZ (6,8) and MPA (11). Studies were conducted to determine the possiblerelationship between these morphologic and biochemical changes and theexpression of genes encoding extracellular matrix molecules (fibronectinand tenascin), receptors for extracellular matrix proteins (α₅ integrinand β₁ integrin), and cytoskeleton proteins (β-actin and γ-actin). Whentreated for 96 h, fibronectin expression was increased under alltreatment protocols, with IFN-β and RA being the least effective agentsin inducing enhanced fibronectin mRNA in HO-1 cells (i.e. signals wereonly detected after long exposures of Northern blots) (FIG. 4 and datanot shown). The most effective single agents resulting in enhancedfibronectin expression were IFN-γ and MPA, whereas MEZ was less potentin inducing enhanced fibronectin expression (FIG. 4). Large increases infibronectin expression were also observed in HO-1 cells grown for 96 hin the combination of IFN-β+IFN-γ, IFN-β+MEZ, MPA+MEZ, and RA+MEZ.IFN-β+MEZ was more effective than MPA+MEZ, RA+MEZ, or a high dose of MEZin inducing enhanced fibronectin expression after 24-h treatment (FIG.5). IFN-β+MEZ also enhanced fibronectin expression to a greater extentthan MPA+MEZ, RA+MEZ, and a high dose of MEZ after removing thiscombination of inducers and growth for 72 h in medium devoid of theinducing agents (FIG. 5).

Expression of the tenascin gene in HO-1 cells treated with the variousdifferentiation-inducing and growth-suppressing agents was more complexthan fibronectin (FIGS. 4 and 5). Growth of HO-1 cells for 96 h in thepresence of IFN-β, MEZ, RA, IFN-β+MEZ, or RA+MEZ resulted in decreasedtenascin expression whereas IFN-γ, MPA, and IFN-β+IFN-α resulted inenhanced tenascin expression (FIG. 4). Ninety-six-hour treatment withIFN-γ, alone or in combination with IFN-β, resulted in the greatestincrease in tenascin expression. In contrast, after a 24-h treatment,tenascin expression was not significantly altered by a high dose of MEZor RA+MEZ, whereas a small increase in tenascin expression was found inIFN-β+MEZ- and MPA+MEZ-treated cultures (FIG. 5). When cultures weretreated for 24 h with inducer and then grown for 72 h in the absence ofinducer, decreased expression of tenascin was observed in high-dose MEZ-and RA+MEZ-treated cultures, whereas only small reductions in tenascinexpression were apparent in MPA+MEZ- or IFN-β+MEZ-treated HO-1 cells(FIG. 5).

Changes were also observed in the expression of matrix receptor genesfor extracellular matrix proteins, α₅ -integrin, β₅ -integrin in HO-1cells grown for (1) 24 h in the presence of the inducer(s), (2) 24 h ininducer(s) followed by 72 h in the absence of inducer(s), or (3)continuously in inducer(s) for 96 h (FIGS. 4 and 5). Increases wereobserved in α₅ integrin expression in HO-1 cells treated for 96 h withIFN-β+IFN-β, IFN-β+MEZ, MPA+MEZ and, to a lesser extent, with RA+MEZ(FIG. 4). Increased α₅ integrin expression was also apparent in HO-1cells treated continuously for 24 hrs or treated for 24 hrs followed by72 hr growth in the absence of inducer(s) to a high dose of MEZ,MPA+MEZ, RA+MEZ, or IFN-β+MEZ. In contrast, α₅ integrin expressed wasreduced in cultures treated with RA for 96 h. In the case of the β₁integrin, upregulation after 96-h treatment was apparent in cellstreated with IFN-γ, IFN-β+MEZ, MPA+MEZ, and RA+MEZ (FIG. 4). The mosteffective inducer of both α₅ and β₁ integrin expression in HO-1 cellswas IFN-β+MEZ (FIGS. 4 and 5). The level of upregulation was greater forα₅ integrin than the β₁ integrin (FIGS. 4 and 5).

The effect of the various growth-suppressing anddifferentiation-modulating compounds on expression of cytoskeletal genes(β-actin and γ-actin) in HO-1 cells is shown in FIGS. 4 and 5. Undermost experimental conditions, only small changes were observed inβ-actin and γ-actin mRNA levels. In the case of 96-h-treated cultures,both β-actin and, to a greater extent, γ-actin expression were decreasedby treatment with a number of agents, resulting in growth suppression.In contrast, RA, which is not growth suppressive in HO-1 cells, did notsignificantly alter the expression of these cytoskeletal genes. A commonchange that was generally most pronounced under all three experimentalprotocols in HO-1 cells, that is, 24-h treatment, 24-h treatmentfollowed by 72-h growth in the absence of inducer, or continuoustreatment for 96 h, was the reduction in β-actin and γ-actin expressionby IFN-β+MEZ.

Modulation of Gene Expression in HO-1 Cells by Conditioned MediumObtained from Differentiation-Inducer-Treated HO-1 Cells.

The studies described previously demonstrated that interferon-responsivegenes and the gro/MGSA gene were activated during the process ofreversible and irreversible differentiation in HO-1 cells. They furthersuggested the possibility of an involvement of autocrine-feedbackpathways in the differentiation process (FIGS. 2 and 3). To determinedirectly if HO-1 cells treated with agents inducing a reversiblecommitment to differentiation (a high dose of MEZ, RA+MEZ, and MPA+MEZ)and/or terminal cell differentiation (IFN-β+MEZ) secrete factor(s) thatcan modulate gene expression in HO-1 cells, conditioned medium wascollected from cells treated for 24 h with the inducer followed bygrowth for 72 h in the absence of inducer (FIGS. 6 and 7). HO-1 cellswere grown in an equal volume of conditioned medium plus and equalvolume of DMEM-10 (1:2) for 24 h or with 1 vol of conditioned mediumplus 3 vol of DMEM-10 (1:4) for 96 h. Total cytoplasmic RNA was thenisolated and analyzed by Northern blotting for the expression of aseries of early growth response, interferon responsive, extracellularmatrix, extracellular matrix receptor, and cytoskeletal genes (FIGS. 6and 7). With the exception of fibronectin and small increases in β₁integrin expression, treatment for 24 h with 1:2 conditioned mediumobtained from the other experimental conditions (which result in areversible commitment to differentiation) did not alter or induceexpression of the genes tested, including c-jun, jun-B, c-myc, gro/MGSA,HLA Class I antigen, ISG-15, α₅ integrin, β-actin, γ-actin, or tenascin.Exposure of HO-1 cells for 96 h to 1:4 conditioned medium obtained fromIFN-β+MEZ-treated HO-1 cells also enhanced fibronectin, HLA Class Iantigen, β₁ integrin and tenascin expression, as well as inducing ISG-15expression. With the exception of fibronectin and tenascin, which wereenhanced by 1:4 conditioned medium obtained from 24-h treated IFN-β+MEZ,MPA+MEZ, RA+MEZ treated cultures, no modification in the expression ofthe various genes was apparent using 1:4 conditioned medium from any ofthe experimental procedures.

The HO-1 human melanoma cell line can be chemically induced toreversibly express specific markers of differentiation or to undergoterminal cell differentiation. The present study was undertaken todetermine which specific programs of gene expression are modified as aconsequence of these cellular alterations. Inductions of terminaldifferentiation, and, to a lesser extent, reversible differentiation,was associated with changes in the expression specific immediate earlyresponse, interferon-responsive, cytokine-responsive, extracellularmatrix, and extracellular matrix receptor genes. In addition,conditioned medium obtained from HO-1 cells treated with IFN-β+MEZ alsoresulted in similar changes in gene expression in naive HO-1 cells asthose observed following direct exposure to the chemical inducers ofterminal differentiation. These results indicate that commongene-expression changes are associated with both the reversible andirreversible induction of differentiation in HO-1 cells. In addition,the terminal differentiation process is correlated with the activationof several autocrine pathways involving both IFN-β and gro/MGSA.

Immediate early response genes, such as c-myc, c-fos, c-jun, jun-B, andjun-D, have been shown to be involved in the regulation of growth and/ordifferentiation in other model systems [33-35]. In the case of c-myc, areduction in expression of this gene is observed in many cell typeseither induced to terminally differentiate or under conditions resultingin a reduction in cellular growth without the induction ofdifferentiation-related genes (36-40). A direct role of c-myc expressionin regulating differentiation in a variety of model cell culture systemshas also been demonstrated using c-myc antisense constructs or oligomers(41-45). In specific systems, the downregulation of c-myc expression byantisense constructs or oligomers has been shown to result indifferentiation and growth suppression in the absence of inducing agents(45-48). Induction of both reversible differentiation and, to a greaterextent, terminal differentiation in HO-1 cells resulted in decreasedc-myc expression. Downregulation of c-myc expression was independent ofgrowth suppression, as indicated by the enhanced expression of c-myc inIFN-β+IFN-γ treated cells, even though this combination of agentsresulted in maximum growth suppression without the induction of anymorphologic or biochemical markers of melanoma differentiation. Based onthe temporal relationship and the magnitude of c-myc downregulation bythe various inducing agents, continued suppression of c-myc expressionmay be required for the induction of terminal differentiation in HO-1cells. Studies using antisense c-myc constructs should prove valuable indirectly addressing the relationship between c-myc expression andterminal differentiation in HO-1 cells.

Two immediate early response genes, c-fos and c-jun, code fortranscription factors involved in nuclear signal transduction (35, 46,47). Expression of these genes can be induced by many external stimuli,including cytokines, growth factors, serum, phorbol esters,neurotransmitters, and viral infection (35, 46, 47). The proteins c-fosand c-jun can form a heterodimer as part of the AP-1transcription-factor complex that binds efficiently to AP-1 sites(TGA^(G) /_(C) TCA) in DNA (35, 46, 47). Previous studies have indicatedthat both the c-jun and c-fos genes are activated during monocyticdifferentiation induced by TPA, macrophage colony-stimulating factor(M-CSF), and okadaic acid (48-50). Elevation of AP-1 activity also hasbeen demonstrated during the induction of differentiation of F9embryonal carcinoma stem cells by RA (51). In contrast thetranscriptional enhancing activity of c-jun, jun-B (which is induced bya number of external stimuli that also induce c-jun) functions as anegative regulator of several genes normally activated by c-jun (52,53). In the process of monocytic differentiation induced by TPA in humancells, jun-B gene transcription, steady-state mRNA levels, and mRNAstability are enhanced (54). Similarly, jun-B expression is enhancedduring the process of monocytic differentiation induced in murine cellsby serum-free conditioned medium from mouse lungs (55). Induction ofgrowth suppression and both reversible and irreversible differentiationin HO-1 cells are unaltered at later time points. Unlike TPA-inducedmonocytic differentiation (48, 54), induction of jun-B expression byIFN-β+MEZ in HO-1 cells is regulated only at the transcriptional level(56). These data indicate that enhanced expressions of c-jun and jun-Bin HO-1 cells is not directly related to the induction of terminaldifferentiation in HO-1 cells. A sustained elevation of c-jun and jun-Bexpression, however, may be components of the differentiation program inHO-1 cells.

The process of cellular differentiation is frequently associated withprofound changes in cellular morphology that are related to cell--celland cell-extracellular matrix interactions as well as the expression ofcell growth and cytoskeletal genes (57). In addition, cell shape andcell-extracellular matrix interactions also play important roles in theprocess of tumorigenesis and metastasis (58, 59). Transformed cellsoften exhibit reductions in fibronectin expression as well as decreasesin expression of specific integrin genes, which encode receptors forextracellular matrix proteins (60, 61). Of particular recent interest isthe α₅ β₁ integrin complex that appears to be the major receptor forfibronectin (62). Decreases in α₅ β₁ expression have been found inoncogenically transformed cells (60) and overexpression of thecombination of α₅ and β₁, integrin cDNA in Chinese hamster ovary (CHO)cells results in a direct suppression of the transformed phenotype (61).Agents that induced reversible differentiation (MPA+MEZ and RA+MEZ),irreversible differentiation (IFN-β+MEZ), and increased growthsuppression without inducing markers of differentiation (IFN-β+IFN-γ) inHO-1 cells enhanced fibronectin, α₅ integrin, and β₁ integrinexpression. These findings suggest that the specific combinations ofcytokines, such as IFN-β+IFN-γ, resulting in growth suppression andcombinations of agents that induce either a reversible commitment todifferentiation or terminal differentiation in HO-1 cells, can directlymodify extracellular matrix and extracellular matrix receptor geneexpression. The changes induced in these genes by these agents reflect amore normal, as opposed to the original, transformed cellular phenotype.In this context, it is also worth commenting on changes induced tenascinexpression as a consequence of treatment with the variousdifferentiation-inducing and/or growth-suppressing agents. Tenascin isan extracellular-matrix protein expressed (or prominently expressed) inspecialized embryonic tissues, cells of neuroectodermal origin, andtumors (63). In general, tenascin is expressed at higher levels inundifferentiated vs. differentiated tumors (63). HO-1 cells expresstenascin, and its level of expression is increased by IFN-β, alone or incombination with IFN-β, whereas its expression is reduced by treatmentwith IFN-β, MEZ, or RA or by continuous growth in the combination ofIFN-β+MEZ or RA+MEZ. These results provide further evidence that thecontinuous treatment of HO-1 cells for 96 h with specificdifferentiation-inducing agents can result in acquisition of a moredifferentiated cellular phenotype by these human melanoma cells.

Studies analyzing the mechanism of growth arrest during the process ofdifferentiation in hematopoietic cells have implicated IFN-β as anautocrine growth inhibitor important in this process (39, 64, 65).Supporting evidence for the involvement of autocrine IFN-β in thedifferentiation process of hematopoietic cells include (1) the abilityof IFN-β antibody, but not IFN-α antibody, to partially block thereduction in c-myc mRNA and growth inhibition associated with thedifferentiation process; (2) the induction of interferon regulatoryfactor 1 (IRF-1), which is a positive transcription factor forexpression of the IFN-β gene, during the myeloid differentiationprocess; (3) the ability of IRF-1 antisense oligomers to partially blockgrowth inhibition associated with IL-6 and leukemia inhibitory factorinduction of differentiation; and (4) the induction of type I interferon(IFN-α/β) gene expression during terminal differentiation inhematopoietic cells (39, 64, 65). A potential IFN-β autocrine loop inthe induction of specific programs of reversible and irreversibledifferentiation in human melanoma cells is also suggested by theexperiments described in this article. Reversible differentiation,resulting from treatment with MPA+MEZ and RA+MEZ, and terminal celldifferentiation, resulting from growth in IFN-β+MEZ, results in theenhanced expression of type I interferon-responsive genes, including HLAClass I antigen, ISG-15, and ISG-54. These same gene expression changesoccur in HO-1 cells treated with conditioned medium obtained fromIFN-β+MEZ treated HO-1 cells. In addition, conditioned medium inducesgrowth suppression in HO-1 cells and IFN-β antibodies partially blockthe induction by conditioned medium of ISG-15 in HO-1 cells (56).Attempts to quantitate IFN-β in conditioned medium form inducer-treatedHO-1 cells have not been successful (66). A possible reason for the lackof quantifiable IFN-β in HO-1 inducer-treated conditioned medium couldbe the presence of INF-β below the sensitivity of detection of the assaysystem, that is, level of IFN-β below 2 U/ml. In this respect, thedifferentiating HO-1 system may be similar to hematopoietic cellsinduced to terminally differentiate by treatment with various inducersthat also produce small quantities of high specific-activity autocrineIFN-β (39). Further studies are required to characterize the putativeautocrine IFN-β produced by differentiating human melanoma cells and todetermine its potential role in both the reversible commitment todifferentiation and terminal differentiation in human melanoma cells.The present studies support the hypothesis that autocrine IFN-β may alsocontribute to the differentiation process in solid tumors.

Analysis of gene expression changes resulting from exposure to IFN-β+MEZand conditioned medium from HO-1 cells treated with these inducerssuggests the presence of additional autocrine factors produced duringthe differentiating process in HO-1 cells. One of these putativeautocrine factors is the previously identified melanoma growth factortermed MGSA (67). MGSA has been identified in the serum-free growthmedium obtained from low-density cultures of the human malignant cellline Hs294T (67). The gene for MGSA has been cloned (68) and the deducedamino acid sequence for human MGSA is identical to that of the human"gro" cDNA isolated by Anisowicz et al. (69), now referred to asgro/MGSA. gro/MGSA is secreted by ˜70% of primary cell cultures derivedfrom human melanoma biopsies and by a majority of benign nevus cellswith chromosomal abnormalities, whereas benign nevus cells with a normalkaryotype are negative for MGSA production (70, 71). The level ofgro/MGSA mRNA is enhanced in human melanoma cells treated with MGSA,indicating a potential autocrine function for this molecule (68); gro-αand gro-β have also been shown to be primary response genes that areinduced as a result of IL-1-mediated growth arrest in human melanomacells (72). In addition, the expression and secretion of MGSA isstrongly induced in other cell types, including human endothelial cellstreated with a number of agents such as IL-1, TNF, lipopolysaccharide,thrombin, or TPA (73). These observations suggest that gro/MGSAproduction is not restricted to human melanoma cells and, in addition tostimulating the growth of specific melanoma cells, gro/MGSA may alsoplay a role in the inflammation process. Applicants presentlydemonstrate that gro/MGSA gene expression is induced in HO-1 cellsduring specific programs of reversible differentiation and duringterminal cell differentiation. In contrast, growth arrest, without theinduction of biochemical or cellular markers of differentiation, doesnot result in gro/MGSA induction. The ability of conditioned mediumobtained from IFN-β+MEZ-treated HO-1 cells to induce gro/MGSA in naiveHO-1 cells suggests that gro/MGSA may be produced during the inductionof terminal cell differentiation in HO-1 cells. At present the functionof gro/MGSA (which is structurally related to a number of additionalgenes, including platelet factor-4, β-thromboglobulin, connectivetissue-activating peptide-3, and the murine KC gene) in melanomadevelopment is not clear. The present data suggest, however, that inaddition to its growth stimulatory effect on human melanoma cells,gro/MGSA may also play a role in melanoma cell differentiation.

In summary, the HO-1 cells culture system has been used to analyze themolecular changes associated with the reversible commitment todifferentiation and terminal cell differentiation in human melanomacells. Evidence is presented indicating that induction of both processesmay involve overlapping gene expression changes. However, the magnitudeof the changes and the persistence of the changes suggest a potentialinvolvement of defined programs of gene expression alterations in theinduction and maintenance of the terminal cells differentiationphenotype of human melanoma cells. Although their precise roles inmelanoma cell growth and differentiation are not presently known, dataare also presented that indicate that induction of differentiationresults in the production of autocrine factors, including IFN-β andgro/MGSA. Further studies are required to define the functionalsignificance of specific gene expression changes and specific autocrinefactors in the process of terminal cells differentiation in humanmelanoma cells. This information will be important in understanding theprocess of melanoma development and evolution and may result in theidentification of novel target genes and molecules that could proveuseful in the therapy of this neoplastic disease. In addition, the HO-1differentiation model system appears ideally suited for theidentification and cloning of genes involved in the induction andmaintenance of loss of proliferative capacity and terminal celldifferentiation in human melanoma cells.

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Second Series of Experiments

Molecular biological approaches for the identification and cloning ofgenes displaying differential expression in both related and differentcell types have been described (1-5). A particularly powerful procedurethat has resulted in the identification of genes differentiallyexpressed in diverse target cells is subtraction hybridization (4,5).This approach has been successfully used to identify genes that arespecifically expressed during progression of the transformed/malignantphenotype (5,6), in cells undergoing growth arrest (7), induced byspecific DNA damaging agents (8), expressed during specific stages of Bcell development (9), and associated with programmed cell death (10).Subtraction hybridization is ideally suited for the identification ofrare transcripts (4,5,9,11,14), or transcripts that exhibit smallvariations in expression between two cell types (4,8,11). As describedin this article, subtraction hybridization is also an ideal procedurefor identifying and cloning genes that are expressed at higher levels incells induced to differentiate versus uninduced parental cells. Inaddition, by performing subtraction in the opposite direction, i.e.,differentiation-inducer treated (Ind⁺) from untreated control (Ind⁻),the present protocol can also be used to identify genes that aresuppressed during terminal differentiation.

Treatment of human melanoma cells with the combination of recombinanthuman fibroblast interferon (IFN-β) and the antileukemic compoundmezerein (MEZ), results in a rapid cessation of cell growth and theinduction of terminal cell differentiation, i.e., cells remain viable,but they lose proliferative capacity (15-17). Terminal celldifferentiation can be induced by IFN-β plus MEZ in human melanoma cellseither innately sensitive or resistant to either agent used alone(15,16). In contrast, treatment of melanoma cells with either IFN-β orMEZ alone results in a reversible alteration in differentiationphenotypes in the human melanoma cell line HO-1. This system representsa valuable experimental model for determining which changes in geneexpression are correlated directly with growth suppression as opposed toreversible differentiation and terminal cell differentiation. Applicantshave presently developed a simple and effective subtractionhybridization protocol and used it to identify melanoma differentiationassociated (mda) genes displaying enhanced expression in cells treatedwith reversible- and terminal differentiation inducing compounds. Fourtypes of mda genes have been identified, including genes upregulated byboth IFN-β and IFN-β plus MEZ, both MEZ and IFN-β plus MEZ, all threetreatments and only the combination of IFN-β plus MEZ. This approachshould prove amenable to other model systems resulting in the isolationof differentially expressed genes involved in important cellularprocesses.

Materials and Methods

Cell Line and Differentiation Induction

The human melanoma cell line HO-1 is a melanotic melanoma derived from a49-year-old female and was used between passage 125 and 150 (16). HO-1cells were grown in Dulbecco's modified Eagle's medium supplemented with10% fetal bovine serum (DMEM-5) at 37° C. in a 5% CO₂ -95% airhumidified incubator. Cells were either untreated (Ind⁻) or treated(Ind⁺) with a combination of IFN-β (2000 units per ml) and MEZ (10 ngper ml) for 2, 4, 8, 12 and 24 hr. For expression studies, HO-1 cellswere untreated or treated for 12 and 24 hr with IFN-β (2000 units perml), MEZ (10 ng per ml) or IFN-β plus MEZ (2000 units per ml plus 10 ngper ml) prior to isolation of cellular RNA and Northern blottinganalysis (17).

Construction of cDNA Libraries

Total cellular RNA from untreated (Ind⁻) and IFN-β plus MEZ treated(2,4,8,12 and 24 hr) (Ind⁺) samples was isolated by the guanidiniumisothiocyanate/CsCl centrifugation procedure and poly(A⁺) RNA wasselected following oligo(dT) cellulose chromatography (18). cDNAsynthesis was performed using the ZAP-cDNA™ synthesis kit fromStratagene® (La Jolla, Calif.) which is based on an adaptation of theGubler and Hoffman method (19). A primer-adapter consisting of oligo(dT)next to a unique restriction site (Xhol) was used for first strandsynthesis. The double-stranded cDNAs were ligated to EcoRI adapters andthen digested with the Xhol restriction endonuclease. The resultantEcoRI and Xhol cohesive ends allowed the finished cDNAs to be insertedinto the λ ZAP II vector in a sense orientation with respect to thelac-Z promoter (20). The λ ZAP II vector contains pBluescript plasmidsequences flanked by bacteriophage-derived f1 sequences that facilitatein vivo conversion of the phage clones into the phagemid (20). Two cDNAlibraries were constructed: a differentiation inducer-treated cDNAlibrary (Ind⁺) (tester library); and a control uninduced cDNA library(Ind⁻) (driver library). The libraries were packaged with Gigapack IIGold Packaging Extract (Stratagene®) and amplified on PLK-F' bacterialcells (Stratagene®).

Preparation of Double-Stranded DNA from Ind⁺ Library

The Ind⁺ cDNA phagemid library -was excised from λ ZAP using the massexcision procedure described by Stratagene® (La Jolla, Calif.) [21].Briefly, 1×10⁷ pfu of Ind⁺ cDNA library were mixed with 2×10⁸ XL-1 Bluestrain of Escherichia coli and 2×10⁸ pfu of ExAssist helper phage in 10mM MgSO₄ followed by absorption at 37° C. for 15 min (22). After theaddition of 10 ml of LB medium, the phage/bacteria mixture was incubatedwith shaking at 37° C. for 2 hr followed by incubation at 70° C. for 20min to heat inactivate the bacteria and the λ ZAP phage particles. Aftercentrifugation at 4000 g for 15 min, the supernatant was transferred toa sterile polystyrene tube, and stored at 4° C. before use.

To produce double-stranded DNA, 5×10⁷ pfu of the phagemids were combinedwith 1×10⁹ SOLR strain of Escherichia coli, which are nonpermissive forthe growth of the helper phages and therefore prevent coinfection by thehelper phages (22), in 10 mM MgSO₄ followed by absorption at 37° C. for15 min. The phagemids/bacteria were transferred to 250 ml LB mediumcontaining 50 μg/ml ampicillin and incubated with shaking at 37° C.overnight. The bacteria were harvested by centrifugation, and thedouble-stranded phagemid DNA was isolated by the alkali lysis method(18) and purified through a QIAGEN-tip 500 column (QIAGEN Inc.,Chatsworth, Calif.).

Preparation of Single-Stranded DNA from Control Ind⁻ Library

The control Ind⁻ cDNA library was excised from lambda ZAP using the massexcision procedure described above. The phagemid (5×10⁷) were combinedwith 1×10⁹ XL-1 Blue strain of Escherichia coli in 10 mM MgSO₄ followedby absorption at 37° C. for 15 min. The phagemids/bacteria weretransferred to 250 ml LB medium, and incubated with shaking at 37° C.for 2 hr. Helper phage VCS M13 (Stratagene®, La Jolla, Calif.) was addedto 2×10⁷ pfu/ml, and after incubation for 1 hr, kanamycin sulfate(Sigma) was added to 70 μg/ml. The bacteria were grown overnight. Thephagemids were harvested and single-stranded DNAs were prepared usingstandard protocols (18).

Pretreatment of Double- and Single-Stranded DNA Prior to Hybridization

To excise the inserts from the vector, double-stranded DNA from the Ind⁺cDNA library was digested with EcoRI and Xhol, and extracted with phenoland chloroform followed by ethanol precipitation (5). Aftercentrifugation, the pellet was resuspended in distilled H₂ O.Single-stranded DNA from Ind⁻ cDNA library was biotinylated usingphotoactivatable biotin (Photobiotin, Sigma, St. Louis, Mo.) (23). In a650 μl microcentrifuge tube, 50 μl of 1 μg/μl single-stranded DNA wasmixed with 50 μl of 1 μg/μl photoactivatable biotin in H₂ O. Thesolution was irradiated with the tube slanted on crushed ice at adistance of 10 cm from a 300 watt sun lamp for 15 min. The DNA wasfurther biotinylated by adding 25 μl of photoactivatable biotin to thesolution which was then exposed to an additional 15 min of irradiationas described above. To remove unlinked biotin, the reaction was dilutedto 200 μl with 100 mM Tris-HCl, 1 mM EDTA, pH 9.0, and extracted 3× with2-butanol. Sodium acetate, pH 6.5 was added to a concentration of 0.3 M,and the biotinylated DNA was precipitated with two volumes of ethanol.

Subtracted Hybridization and Construction of Subtracted cDNA Library

Subtraction hybridization was performed essentially as described byHerfort and Garber (24) with minor modifications. In a 650 μlsiliconized microcentrifuge tube, 400 ng of EcoRI- and Xhol-digestedInd⁺ cDNA library and 12 μg of biotinylated Ind⁻ cDNA library were mixedin 20 μl of 0.5 M NaCl, 0.05 M HEPES, pH 7.6, 0.2% (wt/vol) sodiumdodecyl sulfate and 40% deionized formamide. The mixture was boiled for5 min and incubated at 42° C. for 48 hr. The hybridization mixture wasdiluted to 400 μl with 0.5 M NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA andthen 15 μg of streptavidin (BRL®) in H₂ O was added, followed byincubation at room temperature for 5 min. The sample was extracted 2×with phenol/chloroform (1:1), followed by back-extraction of the organicphase with 50 μl of 0.5 M NaCl in TE buffer, pH 8.0. An additional 10 μgof streptavidin was added and phenol/chloroform extraction was repeated.After removal of excess chloroform by brief lyophilization, the finalsolution was diluted to 2 ml with TE buffer, pH 8.0, and passed througha Centricon 100 filter (Amicron; Danvers, Mass.) 2× as recommended bythe manufacturer. The concentrated DNA solution (approximately 50 μl)was then lyophilized. The subtracted cDNAs were ligated to EcoRI- andXhol-digested and CIAP treated arms of the λZAP II vector and packagedwith Gigapack II Gold packaging extract (Stratagene®, La Jolla, Calif.).The library was then amplified using the PLK-F' bacterial cell.

Screening Subtracted cDNA Library

The mass excision of the library was performed using ExAssist helperphage as described above. The SOLR strain of Escherichia coli and cDNAphagemids were mixed at 37° C. for 15 min and plated onto LB platescontaining ampicillin and IPTG/X-gal. White colonies were chosen atrandom, isolated and grown in LB medium. Plasmid minireps andrestriction enzyme digestions were performed to confirm the presence ofinserts. The inserts were isolated and used as robes for Northernblotting analysis (5,25). Total cellular RNA was prepared from HO-1cells treated with IFN-β (2000 units/ml), MEZ (10 ng/ml), and IFN-β plusMEZ (2000 units/ml plus 10 ng/ml), electrophoresed in 0.8% agarose gelsand transferred to nylon membranes (Amersham, Arlington Heights, Ill.).Radiolabeled probes were generated by random oligonucleotide priming(25). Prehybridization, hybridization, posthybridization washes, andautoradiography were performed as described (5,18,25).

Sequencing of mda Genes

The mda clones were sequenced using double-stranded pBluescript DNA asthe template. DNA sequencing was performed using the Sangerdideoxynucleotide method with sequenase (United States BiochemicalCorp., Cleveland, Ohio) and T3 promoter primer (GIBCO® BRL®,Gaithersburg, Md.). This approach generates sequences from the 5' end ofthe inserts. Sequences were tested for homology to previously identifiedsequences using the GenBank FMBL database and the GCG/FASTA computerprogram.

Experimental Results

Subtraction hybridization represents a valuable methodology forisolating cDNA clones representing preferentially expressed mRNAswithout prior knowledge of the selected gene or its encoded product(1-10). This procedure results in a substantial enrichment ofdifferentially expressed cDNA clones and is often preferable todifferential hybridization procedures using total cDNAs (4,5,11-14). Anumber of protocols have been reported for the generation of subtractionlibraries [reviewed in 4]. The traditional approach involveshybridization of a first strand cDNA (tester) made from the mRNA of onecell type with mRNA (driver) prepared from a second cell type [or thefirst cell type treated with a specific gene modulating agent(s)] [7-9].Single-stranded unhybridized cDNAs are then selected by hydroxylapatitecolumn chromatography and they are used as templates for the synthesisof second-strand cDNA (7-9). However, this procedure has a number oflimitations, including the requirement for RNA handling duringhybridization, which can be problematic, and the limited quantity ofcDNAs recovered following hybridization and column chromatography. Othersubtraction hybridization protocols involve hybridization of cDNA andphotobiotinylated RNA (23,26). Problems may still arise because of therequirement for large amounts of mRNA and from manipulation of RNAduring the hybridization procedure (22). Recent improvements insubtraction hybridization utilize cDNA libraries as both tester anddriver nucleic acid populations (24,27,28). By using driver sequencespresent in cloned forms, the newer approaches circumvent the problemsassociated with insufficient quantities of mRNAs or difficultiesresulting during the preparation and manipulation of mRNAs. Improvementsin subtraction hybridization procedures have included: the use ofphagemid subtraction hybridization (27); the use of single-strandedphagemids with directional inserts (28); and the use of double-strandedcDNA inserts as tester and single-stranded cDNAs as the driver (21).

The procedure applicants have used to construct subtraction librariesinvolves a modification of the protocols described by Rubenstein et al.(28) and Herfort and Garber (24). This strategy is outlined in FIG. 8.Applicants' approach to subtraction library construction uses λ phageand commercially available reagents. In other similar procedures(22,27,28), the end products of subtraction hybridization are eithersingle-stranded phagemid DNA, which is converted to double-stranded DNA,or double-stranded inserts, which are ligated to plasmid vectors. Theseprocedures have two potentially limiting drawbacks including, the lowerefficiency of bacterial transformation with plasmids versus phageinfection and the need for special precautions to remove thedouble-stranded phagemids contaminating the driver single-stranded DNApreparation. By using λ page as vectors, these problems are easilyavoided. The efficiency of phage infection of bacteria is high, oftenattaining levels of 10⁹ PFU/μg DNA (21). In addition, problems withcontaminating plasmids in the preparation are also eliminated becausethey will not be packaged and transfected into bacteria. This approach,therefore, results in the construction of subtraction libraries of hightiter. By employing λ Uni-ZAP vectors which can be converted intophagemids by in vivo excision, the laborious work of subcloning the DNAinserts into plasmids is unnecessary.

cDNA libraries and subtraction libraries are prepared using thecommercial ZAP-cDNA™ synthesis kit from Stratagene® (La Jolla, Calif.)(5). This product has several advantages for the construction ofsubtraction libraries. First, the Xhol adapter-primer permits the cDNAto be inserted into the vector in a unidirectional orientation. Theefficiency of subtraction hybridization will be high if hybridizationoccurs only between complementary molecules in the different cDNAlibraries instead of complementary molecules in the same cDNA library.This improved subtraction hybridization is achieved by using bothsingle-stranded and double-stranded unidirectional cDNA libraries fromeach experimental condition. For construction of mda subtractionlibraries (FIG. 8), both the HO-1 control Ind and the IFN-β plus MEZtreated Ind⁺ cDNA libraries were constructed in a unidirectional manner.The efficiency of subtraction hybridization was insured by usingsingle-stranded unidirectional Ind⁻ cDNA as the driver. Secondly, thebacteriophage f1 origin of replication, which is present in the λ ZAP IIvector, permits excision of pBluescript II SK(-) phagemids from thebacteriophage and rescue of single-stranded DNA with the assistance ofhelper phage (20). The Escherichia coli strains XL-1 blue and SOLR,which are provided as part of the ZAP-cDNA™ kit, are very useful inpreparing single-stranded and double-stranded phagemid DNA. The XL-1blue strain is permissive for ExAssist helper phage growth, while theSOLR strain is nonpermissive (22). The phagemids are excised withExAssist helper phage in XL-1 blue bacterial cells. The phagemids arethen grown in SOLR bacterial cells for harvesting double-stranded DNA orin XL-1 blue cells with helper phages for harvesting single-strandedDNA. Using this approach, contamination of helper phage andsingle-stranded DNA in the double-stranded DNA preparation wasminimized. Contamination could decrease the efficiency of subtractionhybridization because of the complementary binding between thesingle-stranded cDNA and any potentially unique sequences from the samecDNA library (tester). This could potentially result in a failure of theunique sequences to from double-stranded inserts with appropriate endswhich can be ligated into the vectors. A third consideration is thecommercial availability of Uni-ZAP arms which can be used as vector forthe construction of subtraction libraries. Tester inserts are releasedfrom the phagemid vector by digestion with the restriction enzyme EcoRIand Xhol. After subtraction hybridization, the remaining inserts whichare in the double-stranded form because of complementary hybridizationare ligated in a unidirectional manner into Uni-ZAP arms because of theEcoRI and Xhol cohesive ends. This approach eliminates the requirementfor additional vectors. The subtraction library is then converted into aphagemid library which can be easily manipulated for screening,sequencing, in vitro RNA transcription, and mutagenesis. Without theadvent of well designed commercial kits, subtraction hybridization andsubtraction library construction is both time and labor intensive. Theprocedure applicants describe in this article is straight forward andhighly efficient in producing subtraction libraries.

Employing the approaches applicants describe above, cDNA libraries fromcontrol HO-1 cells (Ind⁻) and HO-1 cells treated with the terminaldifferentiation inducing agents IFN-β plus MEZ (Ind⁺) have beenconstructed. The original titers of the cDNA libraries were 1.2×10⁶ PFUand 1.7×10⁶ PFU for the Ind⁻ and Ind⁺, respectively. The high titersobtained suggest that the cDNA libraries are representative of the mRNAsproduced under the experimental conditions used. The purity of thesingle-stranded and double-stranded DNA was examined by digestion withthe restriction endonucleases EcoRI and XhoI. Unlike double-strandedDNA, the single-stranded DNA could not be digested with the restrictionendonucleases. This is demonstrated in FIG. 9, in which plasmid vectoris released after digestion of double-stranded DNA but notsingle-stranded DNA. Four hundred ng of double-stranded DNA(tester:IND⁺) and 12 μg of single-stranded DNA (driver:Ind⁻) were usedfor subtraction hybridization. After a single-round of hybridization,the Ind⁺ subtraction library was constructed in Uni-ZAP XR vector withan original titer of 8 to 10×10³ PFU. Additional rounds of subtractionhybridization resulted in a low percentage of colonies which containedinserts. This may result because of the low concentration of potentiallyunique sequences remaining after the first round of subtractionhybridization. This observation indicates that the subtractionhybridization protocol applicants have utilized is very efficient andthe requirement for additional subtraction hybridizations may not benecessary to identify differentially expressed genes.

Screening of subtraction libraries for differentially expressedsequences can be achieved using several procedures. In a number ofstudies, subtraction libraries are screened using differentialhybridization techniques (7, 8, 27). However, the sensitivity of thisprocedure is limited by the relative abundance of the target mRNA. Theenrichment of target sequences obtained in our subtractions librariespermitted the random isolation of clones for evaluation of mRNAexpression in undifferentiated HO-1 cells or HO-1 cells treated withIFN-β, MEZ or IFN-β+MEZ. After in vivo excision, bacteria containing thesubtraction library were plated and randomly isolated clones were usedto prepare plasmids. The EcoRI/XhoI digested cDNA inserts from theseclones were then used as probes for Northern blotting analysis of mRNAexpression under the different experimental conditions. Among 70 cDNAclones initially analyzed, 23 clones were found to display differencesin gene expression between Ind⁻ and Ind⁺ treated HO-1 cells. Asexpected, subtraction of control HO-1 cDNAs from IFN-β plus MEZ treatedHO-1 cDNAs results in a series of mda genes which displayed enhancedexpression after 24 hr treatment with the inducer. These included mdagenes which were inducible by both IFN-β and IFN-β plus MEZ, i.e. mda-1and mda-2; by both MEZ and IFN-β plus MEZ, i.e., mda-3; by IFN-β, MEZand IFN-β plus MEZ, i.e., mda-4; and uniquely by IFN-β and MEZ, i.e.,mda-5 and mda-6 (FIG. 10). Specific mda genes also displayed elevatedexpression after 96 hr exposure to IFN-β plus MEZ (data not shown).

Of the six mda genes reported in this study, only mda-3 corresponds to apreviously reported gene (FIG. 11). At present, 245 bp of mda-3 havebeen sequenced and this cDNA shares >99% homology with the reportedsequences of pLD78 (29), pAT 464 (30,31), pAT 744 (31) and GOS19(32-34). The pLD78 cDNA is inducible by either TPA or a T-cell mitogen,phytohemagglutinin (PHA), in human tonsilar lymphocytes (29). mda-3 isinduced in HO-1 cells within 24 hr of treatment with MEZ, IFN-β plus MEZand IFN-β plus TPA (data not shown). The sequence of the 5' flankingregion of the genomic DNA encoding for the pLD78 cDNA displayed asignificant homology with corresponding regions of the human interleukin2 and immune interferon genes (29). pAT 464 and pAT 744 are inducible byTPA and PHA, with maximal induction resulting from the combination ofagents, in T-cells, B-cells and the promyelocytic cell line HL-60 (31).In contrast, these cDNAs are not expressed in human fibroblasts,although as indicated in the present study a potentially similar cDNA,mda-3, is inducible in human melanoma. pAT 464 and pAT 744 share somecritical amino acid similarity with a family of secreted factorsincluding connective tissue activating factor III, platelet factor 4, anIFN-γ-induced factor, macrophage inflammatory protein and a factorchemotactic to neutrophils (3-10C, monocyte-derived neutrophilchemotactic factor, neutrophil-activating factor) (31). GOS19 genes aremembers of the "small inducible" family of genes, which exhibit similarexon-intron organizations and which encode secreted proteins withsimilar organization of cysteine and proline residues (32-34). TheGOS19-1 -mRNA is enhanced rapidly by the addition of both cycloheximideor lectin to cultured human blood mononuclear cells (32). This cDNA hassequence homology to the murine gene that encodes an inhibitory cytokine(MIPIα/SCI) which decreases stem cell proliferation (32). In thiscontext, GOS19-1, which is the main GOS19 gene expressed in adult Tlymphocytes, may encode a homeostatic negative regulator of marrow stemcell populations. The role of mda-3 in the process of melanoma cellgrowth and differentiation remains to be determined.

Studies are currently in progress to further characterize the novel mdagenes, mda-1, mda-2, mda-4, mda-5, and mda-6, and determined theirexpression in different stages of melanoma evolution and during theinduction of growth suppression, the reversible commitment todifferentiation and the induction of terminal differentiation in humanmelanoma cells. It should be emphasized that the cDNA clones applicantshave currently analyzed represent only a small percentage of thecomplete subtraction library. This suggests that this subtractionlibrary has the potential for identifying and cloning additional genesinvolved or associated with the chemical induction of differentiationand growth suppression in human melanoma cells. In addition, by alteringthe driver DNA, i.e., using combinations of cDNA libraries constructedfrom HO-1 cells treated singularly with IFN-β and MEZ, it should bepossible to further enrich for gene(s) uniquely expressed in terminallydifferentiated human melanoma cells, i.e. those treated with thecombinations of IFN-β+MEZ.

In summary, applicants presently describe an efficient and sensitiveprocedure for the production of subtraction hybridized cDNA librarieswhich can be used for the identification and cloning of differentiallyexpressed genes. The basic protocol utilizes biotinylatedsingle-stranded DNA as the driver and bacteriophage as the vector andrelies on the availability of commercial reagents for construction ofsubtraction cDNA libraries. The usefulness of the current protocol isdemonstrated by the high level of enrichment obtained from genes in thesubtracted library associated with the induction of differentiation ofhuman melanoma cells, i.e., mda genes. This procedure should find wideapplicability for the identification and cloning of differentiallyexpressed genes. These can include, but are not limited to, genesdisplaying modified expression between closely related cell types,between disparate cell types, in cells induced to lose proliferativeability or undergo apoptosis, in cells treated with chemotherapeuticagents, and in cells induced or committed to reversible or terminaldifferentiation.

References for the Second Series of Experiments

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3. Lee, S. W., Tomasetto, C., and Sager, R. Positive selection ofcandidate tumor-suppressor genes by subtractive hybridization, Proc.Natl. Acad. Sci. USA, 88:2825-2829 (1991).

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5. Reddy, P. G., Su, Z.-Z., and Fisher, P. B. Identification and cloningof genes involved in progression of transformed phenotype. In: Adolph,K. W., Ed., Methods in Molecular Genetics, Vol. I, Academic Press,Orlando, Fla., pp. 68-102 (1993).

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10. Owens, G. P., Hahn, W. E, and Cohen, J. J. Identification of mRNAsassociated with programmed cell death in immature thymocytes, Mol. Cell.Biol., 11:4177-4188 (1991).

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12. Frohman, M. A., Dush, M. K., and Martin, G. R. Rapid production offull-length cDNAs from rare transcripts: amplification using asingle-gene specific oligonucleotide primer, Proc. Natl. Acad. Sci. USA,85:8998-9002 (1988).

13. Timblin, C. J., Battey, G., and Kuehl, W. M. Application for PCRtechnology to subtractive cDNA cloning: Identification of genesexpressed specifically in murine plasmacytoma cells, Nucleic Acids Res.,18:1587-1593 (1990).

14. Wieland, I., Bolgar, G., Asouline, G., and Wigler, M. A method fordifference cloning: Gene amplification following subtractivehybridization, Proc. Natl. Acad. Sci. USA, 87:2720-2724 (1990).

15. Fisher, P. B., Hermo, H., Jr., Solowey, W. E., Dietrich, M. C.,Edwalds, G. M., Weinstein, I. B., Langer, J. A., Pestka, S., Giacomini,P., Kusama, M., and Ferrone, S. Effect of recombinant human fibroblastinterferon and mezerein on growth, differentiation, immune interferonbinding and tumor associated antigen expression in human melanoma cells,Anticancer Res., 6:765-774 (1986).

16. Fisher, P. B., Prignoli, D. R., Hermo, H., Jr., Weinstein, I. B.,and Pestka, S. Effects of combined treatment with interferon andmezerein on melanogenesis and growth in human melanoma cells, J.Interferon Res., 5: 11-22 (1985).

17. Jiang, H., Su, Z.-Z., Boyd, J., and Fisher, P. B. Gene expressionchanges induced in human melanoma cells undergoing reversible growthsuppression and terminal cell differentiation, Mol. Cell. Different.,1:41-66 (1993).

18. Sambrook, J., Fritsch, E. F., and Maniatis, T., Eds., MolecularCloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Lab.Press, Cold Springer Harbor, N.Y., 1989.

19. Gubler, U., and Hoffman, B. J. A simple and very effective methodfor generating cDNA libraries, Gene, 25:263-269 (1983).

20. Short, J. M., Fernandez, J. M., Sorge, J. A., and Huse, W. D. λ ZAP:A bacteriophage λ expression vector with in vivo excision properties,Nuc. Acids Res., 16:7583-7600 (1988).

21. Short, J. M., and Sorge, J. A. In vivo excision properties ofbacteriophage λ ZAP expression vector, Meth. Enz., 216:495-508 (1992).

22. Hay, B., and Short, J. M. ExAssis™ helper phage and SOLR™ for lambdaZAP II excision, Stratagies, 5:16-18 (1992).

23. Sive, H. L., and St. John, T. A simple subtraction techniqueemploying photoactivable biotin and phenol extraction, Nuc. Acids Res.,16:10937 (1988).

24. Herfort, M. R., and Garber, A. T. Simple and Efficient subtractivehybridization screening, BioTechniques, 11(5):598-603 (1991).

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26. Swaroop, A., Xu, J., Agarwal, N., and Weissman, S. A simple andefficient cDNA subtraction procedure: isolation of human retina-specificcDNA clones, Nuc. Acids Res., 19:1954 (1991).

27. Duguid, J. R., Rohwer, R. G., and Seed, B. Isolation of cDNAs ofscrapie-modulated RNAs by subtractive hybridization, Proc. Natl. Acad.Sci. USA, 85:5738-5742 (1988).

28. Rubenstein, J. L. R., Brice, A. E. J., Claranello, R. D., Denney,D., Proteus, M. H., and Usdin, T. B. Subtractive hybridization systemusing single-stranded phagemids with directional inserts, Nuc. AcidsRes., 18:4833-4842 (1990).

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31. Zipfel, P. F., Blake, J., Irving, S. G., Kelly, K., and Siebenlist,U. Mitogenic activation of human T cells induces two closely relatedgenes which share structural similarities with a new family of secretedfactors, J. Immunol., 142:1582-1590 (1989).

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Third Series of Experiments

Development of malignant melanoma in humans, with the exception ofnodular type melanoma, is a progressive process involving a discreteseries of well defined stages. Although the focus of intensivescientific scrutiny, the genetic elements controlling melanocyticconversion into the various stages of the evolving melanoma have notbeen identified. In addition, no consistently effective therapy iscurrently available to treat metastatic melanoma. The long-term goal ofthe present proposal is to define the genes regulating melanoma growth,differentiation and progression. This information could prove valuablein elucidating potential targets for therapeutic intervention.

Tumor progression in melanocytes is associated with altered patterns ofnormal melanocytic differentiation. Chemical induction of terminaldifferentiation in tumor cells represents a useful approach forreversing the negative prognosis associated with specific neoplasms.Recent studies indicate that the specific combination of recombinanthuman fibroblast interferon (IFN-β) and the antileukemic compoundmezerein (MEZ) can reprogram human melanoma cells to undergo terminaldifferentiation, i.e., cells retain viability but they irreversibly loseproliferative capacity. In contrast, application of comparable doses ofIFN-β or MEZ alone to human melanoma cells results in a reversiblecommitment to differentiation, i.e., removal of the inducing agentresults in the resumption of cell growth and the loss of specificdifferentiation-associated properties.

Subtraction hybridization is used to identify the genotypic changesassociated with induction of terminal differentiation in human melanomacells. Using this approach, cDNAs displaying enhanced expression inmelanoma cells induced to terminally differentiate versus untreatedmelanoma cells have been identified. Partial sequence analysis of thesedifferentially expressed cDNAs, tentatively called melanomadifferentiation associated (mda) genes, indicate that they consist ofboth known and previously unidentified genes. Specific mda genes mayrepresent novel genetic elements involved in tumor cell growth and/orcommitment of cells to the melanocyte lineage.

The specific aims of this proposal are to characterize and determine thefunctional roles of the mda genes in melanoma growth, differentiation,and progression. With these aims in mind studies will be conducted to:

1) Determine the pattern and regulation of expression of the mda genesin melanocytes, nevi, radial growth phase melanoma, vertical growthphase melanoma and metastatic melanoma cells;

2) Analyze the relationship between mda gene expression and theinduction of reversible commitment to differentiation, growthsuppression without the induction of differentiation, DNA damage andstress responses and induction of terminal differentiation in humanmelanoma and other model differentiation systems;

3) Isolate full-length cDNAs of mda genes that may be involved inmelanoma differentiation or progression and directly determine theirpotential functional role in differentiation and progression of humanmelanoma;

4) Isolate and characterize the promoter region of appropriate mda genesand analyze their regulation in human melanocytes, nevi and melanoma.

Experimental strategies designed to activate genes mediating a loss ofproliferative capacity and the reprogramming of melanoma cells toterminally differentiate, may represent novel approaches for effectivetherapeutic intervention in metastatic melanoma. Elucidation of thefunction of the cloned mda genes should provide molecular insights intothe process of melanoma differentiation and progression. In addition,specific mda genes may represent targets of clinical interest which canbe exploited for suppressing the growth of metastatic melanoma and othertumorigenic cell types.

Background and Significance

Malignant melanoma epitomizes the process of tumor progression andemphasizes the selective nature of the metastatic phenotype and thegrowth dominant properties of metastatic cells (rev. 1 to 3). Of thenumerous types of cancer developing in North American populations,melanoma is increasing at the fastest rate and it is estimated that asmany as 1 in 100 currently born children may eventually developsuperficial spreading type melanoma (3,4). Although melanoma is readilycurable at early stages, surgical and chemotherapeutic interventions arevirtually ineffective in preventing metastatic disease and death inpatients with advanced stages of malignant melanoma. These observationsemphasize the need for improved therapeutic approaches to moreefficaciously treat patients with metastatic melanoma.

Development of malignant melanoma in humans, with the exception ofnodular type melanoma, consists of a series of sequential alterations inthe evolving tumor cells (rev. 1-4). These include conversion of anormal melanocyte into a common acquired melanocytic nevus (mole),followed by the development of a dysplastic nevus, a radial growth phase(RGP) primary melanoma, a vertical growth phase (VGP) primary melanomaand ultimately a metastatic melanoma. As indicated above, althoughreadily treatable during the early stages of development even during theVGP if the lesion is ≦0.76-mm thick, currently employed techniques arenot very effective (<20% survival) in preventing metastatic spread andmorbidity in patients with VGP lesions >4.0-mm thickness. Thisexperimental model system is ideally suited to evaluate the criticalgenetic changes that mediate both the early and late phases of melanomaevolution.

A less toxic approach to cancer therapy involves a process termeddifferentiation therapy (5-9). Two premises underlie this therapeuticmodality. (A) Many neoplastic cells display aberrant patterns ofdifferentiation resulting in unrestrained growth; and (B) Treatment withthe appropriate agent(s) can result in the reprogramming of tumor cellsto lose proliferative capacity and become terminally differentiated.Intrinsic in this hypothesis is the assumption that the genes thatmediate normal differentiation in many tumor cells are not geneticallydefective, but rather they fail to be appropriately expressed. Thesuccessful application of differentiation therapy in specific instancesmay result because the appropriate genes inducing the differentiatedphenotype become transcriptionally activated resulting in the productionof appropriate gene products required to induce terminal celldifferentiation. Applicants have tested this hypothesis using humanmelanoma cells (10-14). Treatment of human melanoma cells with thecombination of recombinant human fibroblast interferon (IFN-β) and theantileukemic compound mezerein (MEZ) results in a rapid cessation ofgrowth, an induction of morphological changes, an alteration inantigenic phenotype, an increase in melanin synthesis and anirreversible loss in proliferative capacity, i.e., terminal celldifferentiation (10,11,14). IFN-β plus MEZ effectively induce terminaldifferentiation in human melanoma cells innately resistant to theantiproliferative effect of either agent used alone (10). In contrast,IFN-β or MEZ applied alone induce a number of similar biochemical andcellular changes in human melanoma cells, however, these changes areoften reversible following removal of the inducing agent, i.e.,reversible commitment to differentiation (10,14). Although the effect ofIFN-β plus MEZ toward normal human melanocytes has not been reported,Krasagakis et al. (15) did determine the effect of IFN-β plus TPA (whichwas present in the melanocyte growth medium) on the growth of normalhuman melanocytes. MEZ shares a number of in vitro properties with TPA,including its ability to replace TPA for the growth of normalmelanocytes, to activate protein kinase C and to modulate celldifferentiation (14,16). In contrast to TPA, however, MEZ is a very weaktumor promoter when substituted for TPA in the initiation-promotionmodel of carcinogenesis on mouse skin, although it is quite potentduring the second phase of tumor promotion (17,18). When normalmelanocytes are grown under optimal growth conditions, including TPA,cholera toxin, isobutylmethylxanthine and fetal bovine serum, even highdoses (10,000 units/ml) of leukocyte (IFN-α), fibroblast (IFN-β) orimmune (IFN-γ) interferon does not inhibit growth (15). In contrast,when grown in modified melanocyte medium not containing TPA andresulting in reduced growth potential, only IFN-β significantly inhibitsproliferation. When tested in serum-free medium, all three interferonpreparations are growth inhibitory toward the SKMel-28 human melanomacell line, with IFN-β again being the most growth-suppressive (15).IFN-β has been shown to be more growth suppressive than IFN-α towardseveral additional human melanomas grown in serum containing medium(10). These results support the hypothesis that IFN-β may be anegative-regulator of melanocyte proliferation and malignanttransformation results in an increased sensitivity to interferons(10,15). In the case of TPA, it is an obligatory requirement for the invitro growth of normal melanocytes, whereas TPA and MEZ are growthinhibitory toward many human melanoma cells (14-16,19,20).

Melanoma represents a useful experimental model to analyze the processof tumor progression (rev 1-3). Cell culture systems are available thatpermit the growth of normal melanocytes, nevi and melanoma cellsrepresenting different stages of tumor progression (1-3,20-25). Analysesof the properties of cells of the melanocyte lineage indicate a numberof traits that allow the different stages in melanoma evolution to bedistinguished. These include: (a) morphology,; (b) life span in culture;(c) chromosomal abnormalities; (d) anchorage-independent growth; (e)tumorigenicity; (f) expression of HLA-DR (class II HLA antigens) andintercellular adhesion molecule-1 (ICAM-1) antigens; (g) response to thetumor promoting agent 12-O-tetradecanoyl-phorbol-13-acetate (TPA); (h)growth factor independence in vitro; (i) autocrine production of basicfibroblast growth factor (bFGF) and (j) growth inhibition by cytokines(rev 2,21,25). A limitation of the melanoma progression model, however,is the inability to obtain from the same patient who has developed aprimary RGP or an early VGP melanoma (less than 0.76 mm in thickness), agenetically related more progressed melanoma. Recent studies by Dr.Kerbel and colleagues (25) suggest that by appropriate manipulation (useof matrigel) and tumor selection in nude mice, it may be possible tospontaneously progress early-stage, non- or poorly tumorigenic (in nudemice) human melanoma cell lines to a more progressed tumorigenic andmetastatic state. In addition, Dr. Albino and colleagues (20)demonstrated that normal human melanocytes could be progressed to acomplete melanoma phenotype and genotype following infection with aretrovirus containing the viral Ha-ras oncogene. Transformed melanocytesacquired the full spectrum of melanoma properties and displayed the samecytogenetic changes occurring during melanoma development in vivo (20).These cell lines will prove useful for evaluating the biochemical andgenetic changes involved in melanoma progression. In summary, theability to clearly define specific components of melanoma evolution willprovide a valuable experimental model to define the genotypic changesmediating tumor progression.

The critical genomic changes that mediate melanoma development andprogression remain to be defined. Recent studies have addressed thepotential relationship between the expression of specific oncogenes,growth factor genes (in addition to basic fibroblast growth factor(bFGF)), growth factor receptor genes, protease genes and early responsegenes and melanoma progression (26-30). Using a panel of metastaticmelanoma cell lines, steady state mRNA transcripts for several growthfactors (bFGF, platelet-derived growth factor (PDGF)-A, PDGF-B,transforming growth factor (TGF)-β₁, TGF-α, melanoma growth-stimulatingactivity (MGSA; also called gro), interleukin (IL-1α and IL-1β) andearly response (c-fos, c-jun and jun-B) genes have been found (27, 28).All of the metastatic melanoma cell lines expressed the bFGF gene andthe majority of metastatic melanoma expressed c-fos, c-jun and jun-B inboth serum-free and serum containing medium. With respect to the othergrowth factor genes tested, each metastatic melanoma displayed a patternof expression that was specific and different (27). In contrast, twostrains of normal melanocytes expressed TGF-β₁ but not bFGF, PDGF, TGF-αor MGSA mRNA at detectable levels (27). Although metastatic melanoma andnormal melanocytes express c-fos, c-jun and jun-B, the expression ofthese transcripts in normal melanocytes was dependent on the presence ofgrowth-promoting agents in the medium (28). In contrast, differentlevels of the early response genes were observed in metastatic melanomacells grown in the presence or absence serum (28). In general, anincrease in jun-B and c-fos RNA transcripts and a decrease in c-jun RNAtranscripts were observed in metastatic melanomas compared to neonatalmelanocytes (28). The relevance of these differences in early responsegene expression in metastatic melanomas compared to neonatal melanocytesremains to be determined. In a recent study, Albino et al. (30) used PCRto determine the level of RNA transcripts for 11 different growthfactors in 19 metastatic human melanoma cell lines and 14 normal humanforeskin melanocyte cell lines. Transcripts for TGF-β₂ (19 of 19), TGF-α(18 of 19) and bFGF (19 of 19) were found in metastatic melanoma but notin the normal melanocytes. In contrast, TGF-β₁ and TGF-β₃ were expressedin both metastatic melanoma and normal melanocytes. The significance ofthese changes to melanoma progression is not apparent. These resultssuggest, however, that the differential expression of specific genes,i.e., bFGF, TGF-β₂, TGF-α and possibly early response genes, maycontribute to or may be directly related to the metastatic melanomaphenotype.

On the basis of genetic linkage analysis of familial melanoma,cytogenetic analysis, and various molecular techniques (including RFLPanalysis to identify LOH in tumor DNA samples and microcell genetransfer procedures) it is now apparent that nonrandom changes in geneson chromosomes 1, 6, 7 and 9 may contribute to the etiology of humanmelanoma (31-41). At this stage of analysis, at least 5 genes, mappingto chromosomes 1, 6, 7 and 9, appear to contribute to the development ofmalignant melanoma, and extensive tumor heterogeneity also implicatesadditional loci as contributors to the malignant phenotype (rev. 38,41).A proposed model of tumor progression from melanocyte to metastaticmelanoma suggests that alterations in chromosome 1 and 9 are earlyevents in melanoma progression, whereas changes in chromosome 6 and 7represent later stages of tumor progression (38,41). A directdemonstration of the suppressive role of chromosome 6 in human melanomahas recently been demonstrated (37). Employing microcell mediated genetransfer, a normal human chromosome 6 was inserted into human melanomacells (UACC-903) and shown to suppress transformed properties in vitroand tumorigenic potential in nude mice (37). Dr. Welch and colleagueshave also demonstrated that insertion of a normal human chromosome 6into the C8161 human melanoma cell line results in a suppression ofmetastatic potential, but not tumorigenic potential (42). The apparentdiscrepancy between the results of Trent et al. (37) and Welch et al.(42) may relate to differences between UACC-903 and C8161 cells. C8161cells exhibit both tumorigenic and metastatic properties in nude mice,whereas UACC-903 cells are tumorigenic but not metastatic in nude mice.This difference might mask the presence of a metastasis suppressor onchromosome 6 or alternatively might suggest that chromosome 6 containsboth a tumor and a metastatic suppressor gene. The chromosome 6containing C8161 cells have also been found to differ from parentalC8161 cells in their biological response and in gene expression aftertreatment with IFN-β plus MEZ (43). Further studies are required todetermine if a similar suppression of transformed and tumorigenicproperties can be induced by reintroduction by microcell mediatedtransfer of chromosomes 1, 7 and/or 9 into melanoma cells containingabnormalities in these chromosomes. Similarly, the mechanism by whichthe putative melanoma suppressor gene(s) on chromosome 6, as well assuppressor gene(s) located on additional chromosomes, exert theireffects on human melanoma cells and how these genes regulate tumorprogression remain to be determined.

Recent studies have demonstrated the existence of at least 23 IFN-αgenes and pseudogenes, all of which reside proximal to the IFN-β genethat is located on locus 9p (9p22-13) (44,45). Nonrandom alterations inspecific loci on chromosome 9 appear to be an early event in melanomaevolution. It is intriguing, therefore, that 90% (9/10) of informativemelanoma DNAs have shown a reduction for one to five of the loci testedin the same region as the IFN-α/β gene (41) including a 2 to 3 megabaseregion on 9p21 in which a putative melanoma tumor-suppressor geneappears to be located (40). Similarly, homo- or homozygously deleted α-and β-interferon genes have been found in human acute lymphoblasticleukemias and human malignant gliomas (44-47). This observation isinteresting, since interferons display antiproliferative activity towardboth human melanoma and lymphoblastic leukemic cells and can be viewedtherefore as tumor suppressor proteins (rev. 13). This data iscompatible with the hypothesis that a tumor suppressor locus for bothmelanoma and leukemia is located on chromosome 9 and tumor suppressionmay in specific cancers involve alterations in the interferon generegion.

The mechanism by which the combination of IFN-β+MEZ induces a rapidirreversible inhibition in cellular proliferation and terminaldifferentiation in human melanoma cells remains to be determined. Sinceactinomycin D and cycloheximide can inhibit the induction of morphologicchanges, growth suppression and the induction of differentiation in HO-1cells induced by IFN-β+MEZ (49), transcriptional activation orsuppression of specific gene(s) following treatment with these agentsmay be the primary determinants of induction of differentiation. Amodified subtraction hybridization procedure was used to identify andcharacterize the critical genes that mediate and which are associatedwith the chemical induction (49). Using this approach a series of cDNAshave been identified, termed melanoma differentiation associated (mda)genes, which display enhanced expression in terminally differentiatedhuman melanoma cells (49). Specific cDNAs have been identified whichrepresent novel genes, i.e., their sequences have not been describedpreviously in any of the DNA data bases. By using appropriate sense andanti-sense oligomers and expression constructs, studies will beconducted to determine the functional role of the mda genes in melanomagrowth, differentiation and progression. In addition, by employing humanmelanoma cells representing specific stages in melanocytic evolution tometastatic melanoma it will also be possible to address the relationshipbetween states of tumor progression and susceptibility to induction ofterminal differentiation. An understanding of the process of terminalcell differentiation and the function of the mda genes could proveuseful in defining the molecular basis of melanoma progression and indesigning improved strategies for the therapy of malignant melanoma andother cancers.

A. Induction of Terminal Differentiation in Human Melanoma Cells byIFN-β plus MEZ.

As discussed in Background and Significance, a hallmark of many cancersis an inability to undergo normal programs of cellular differentiation.If this assumption is correct, and if the genetic machinery of the tumorcells could be reprogrammed to regain their commitment to normaldifferentiation, then appropriate external stimuli could be employed toinduce a loss of proliferative capacity and terminal differentiation(5-14). In studies designed to directly test this hypothesis, applicantshave successfully induced terminal differentiation in human melanomacells with the combination of recombinant IFN-β and the antileukemiccompound mezerein (MEZ) (10,11,14). In contrast, the combination ofrecombinant leukocyte interferon (IFN-α) and MEZ resulted in apotentiation of growth suppression, but terminal differentiation was notinduced, i.e., treated cells retained proliferative capacity (10). Thecombination of IFN-β plus MEZ was effective in inducing terminaldifferentiation in human melanoma cells relatively resistant orsensitive to the growth suppressive effects of either agent employedalone (10). Induction of terminal differentiation in the human melanomacell line HO-1 by continuous exposure for 4 or 7 days to IFN-β plus MEZwas associated with: (a) a rapid, within 24 hr, inhibition inproliferation (FIG. 12) (10,11); (b) a profound alteration in cellularmorphology (treated cells displayed dendrite-like processes) (FIG. 13)(10); and (c) an induction (in melanotic melanoma) or an increasedsynthesis (in melanotic melanoma) of melanin, a marker of melanoma celldifferentiation (10). By employing varying doses of IFN-β and MEZ anddifferent treatment schedules (24 hr, 4 days and/or 7 days), it has beenpossible to separate the chemical-induction of melanoma differentiationinto three stages. These include an early completelyreversible-induction phase (low doses of inducing agents for 4 or 7days), a late partially reversible-induction phase (higher doses ofinducing agents for 4 days), and an irreversibleterminal-differentiation phase (specific doses of inducing agents for 24hr, 4 days or 7 days)) (10-14).

In contrast to IFN-β plus MEZ that induces an irreversible lossin-proliferative capacity and terminal differentiation in the humanmelanoma cell line HO-1, the combination of IFN-β plus IFN-γ inducesenhanced growth suppression without terminal differentiation (12, 14,50). In addition, IFN-β plus IFN-γ also fail to induce an increase inmelanin synthesis in HO-1 cells (50).

When treated with trans retinoic acid (RA), both melanin levels andtyrosinase levels are increased in HO-1 cells, but growth is notsuppressed (14,51). Exposure to 3 μM mycophenolic acid (MPA) for 96 hrresults in growth inhibition, morphologic changes, enhanced melaninsynthesis and enhanced tyrosinase activity in HO-1 cells (14, 51).However, these affects are reversible when HO-1 cells treated with 3 μMMPA for 4 days are then grown in the absence of MPA for an additional 7days (14). These results suggest that at the dose- and time-intervalused, MPA (alone or in combination with MEZ) induces areversible-induction of differentiation in HO-1 cells and not terminaldifferentiation. The effect of different agents on growth and theproperties of HO-1 cells is summarized in Table 2 (14).

                  TABLE 2                                                         ______________________________________                                        Experimental                                                                             Morphology   Melanin  Tyrosinase                                     Conditions.sup.a changes.sup.b Synthesis.sup.c activity.sup.d               ______________________________________                                          RA (2.5 μM) - 1+ 2+                                                        MPA (3.0 μM) + 2+ 3+                                                       MEZ + 1+ NT                                                                   (10 ng/ml)                                                                    IFN-β - 1+ NT                                                            (2000 U/ml)                                                                   IFN-γ - - NT                                                            (2000 U/ml)                                                                   RA + MEZ + NT NT                                                              (2.5 μM +                                                                  10 ng/ml)                                                                     MPA + MEZ + NT NT                                                             (3.0 μM +                                                                  10 ng/ml)                                                                     IFN-β + IFN-γ - 1+ NT                                              (1000 U/ml +                                                                  1000 U/ml)                                                                    IFN-β + MEZ + 4+ NT                                                      (2000 U/ml +                                                                  10 ng/ml)                                                                   ______________________________________                                                      Growth                                                            Experimental Suppression Terminal cell                                        conditions.sup.a (reversible).sup.e differentiation.sup.f                   ______________________________________                                          RA (2.5 μM) - -                                                            MPA (3.0 μM) - -                                                           MEZ 1+ -                                                                      (10 ng/ml)                                                                    IFN-β 3+ -                                                               (2000 U/ml)                                                                   IFN-γ 2+ -                                                              (2000 U/ml)                                                                   RA + MEZ 1+ -                                                                 (2.5 μM +                                                                  10 ng/ml)                                                                     MPA + MEZ 3+ -                                                                (3.0 μM +                                                                  10 ng/ml)                                                                     IFN-β + IFN-γ 4+ -                                                 (1000 U/ml +                                                                  1000 U/ml)                                                                    IFN-β + MEZ  4+.sup.g +                                                  (2000 U/ml +                                                                  10 ng/ml)                                                                   ______________________________________                                         .sup.a HO1 cells were grown for 96 hr or for 6 or 7 days (with medium         changes after 3 or 4 days) in the presence of the agents indicated. For       morphology, cells grown for 96 hr in the test agent were observed             microscopically. For melanin synthesis, results are for 6 day assays for      RA and MPA (51) or 7 day assays for MEZ, IFNβ, IFNγ, IFNβ     + IFNγ and IFNβ + MEZ (10,50). For tyrosinase assays, results      are for 6 day assays for RA, MPA and MEZ (51).  # Growth suppression          (reversible and terminal cell differentiation) assays, refer to cultures      treated with the indicated compound(s) for 96 hr prior to cell number         determination, or treated for 96 hr and then grown for 2 weeks (with          medium changes every 4 days) in the absence of compound prior to cell         number determination.                                                         .sup.b Morphology changes refer to the development of dendritelike            processes 96 hr after growth in the indicated compound. + = presence of       dendritelike processes; - = no dendritelike processes.                        .sup.c Melanin assays were determined as described in refs. 10,50,51.         Results are expressed as relative increases based on separate data            presented in refs. 10,50,51. N.T. = not tested.                               .sup.d Tyrosinase assays were performed as described in ref. 11. Relative     increases (of a similar magnitude) were found for RA, MPA and MEZ after 6     days exposure to these agents (51). N.T. = not tested.                        .sup.e Reversible growth suppression indicates resumption of cell growth      after treatment with the indicated compound(s) for 96 hr, removal of the      test agent and growth for 14 days in compound(s) free medium. Further         details can be found in ref. 14. The degree of initial 96 hr growth           suppression is indicated as: - = no significant change in growth (<10%        reduction in growth in comparison with untreated control cultures); 1+ =      ˜30%  # reduction in growth in comparison with untreated control        cultures; 2+ = ˜40% reduction in growth in comparison with untreate     control cultures; 3+ = ˜50 to 60% reduction in growth in comparison     with untreated control cultures; 4+ = ˜80% reduction in growth in       comparison with untreated control cultures.                                   .sup.f The combination of IFNβ + MEZ results in irreversible growth      suppression.                                                                  .sup.g Terminal cell differentiation indicates the loss of proliferative      capacity after treatment with the indicated compound(s) for 96 hr, remova     of the test agent and growth for 14 days in compound(s) free medium.          Further details can be found in (14).                                    

The studies briefly described above indicate that changes in growth,morphology, melanin synthesis and tyrosinase activity can be dissociatedfrom the induction of terminal differentiation in HO-1 melanoma cells.However, the irreversible loss of proliferative capacity and terminaldifferentiation resulting from treatment with IFN-β plus MEZ appear tobe correlated phenomena. Employing the various agents described above itwill be possible to determine which gene expression changes are relatedto the various components of the differentiation process in humanmelanoma cells.

Monoclonal antibodies (MAbs) have recently been developed whichrecognize a series of hnRNP proteins, designated P2Ps, which display amarked reduction in both 3T3T cells and human keratinocytes induced toterminally differentiate (52,53). In contrast, P2Ps are present in cellsthat have retained the ability to traverse the cell cycle, includingcells reversibly growth arrested. A loss of P2Ps is also observed incells that have irreversibly lost proliferative potential as aconsequence of senescence, as well as induction of terminaldifferentiation (52,53). In contrast, 3T3T cells transformed by SV40 donot undergo the terminal step of differentiation and these cells also donot show a suppression of P2P expression (52). These results support theconcept that P2Ps may be directly linked to proliferative capacity ofcells and may prove useful as a general marker for terminal celldifferentiation. In collaboration with Dr. Robert E. Scott (Universityof Tennessee Medical Center, Memphis, Tenn.) applicants have begun todetermine the level of P2Ps in human melanoma cells induced toterminally differentiate by exposure to IFN-β plus MEZ and MPA plus MEZ(FIG. 14). When induced to terminally differentiate, a reduction in P2Pswas apparent in several independent human melanoma cell lines (data fromFO-1 human melanoma cells is shown in FIG. 14). In contrast, employinghuman melanocytes immortalized by the SV40 T-antigen gene (54), IFN-βplus MEZ did not induce terminal differentiation or a reduction in P2Ps,whereas MPA plus MEZ resulted in a loss of proliferative capacity and areduction in P2Ps. In both FO-1 and FM516 SV cells, treatment withIFN-β, MEZ or MPA alone did not reduce P2Ps even though growth wassuppressed (data not shown). Induction of terminal differentiation byIFN-β+MEZ in HO-1 cells resulted in a loss of P2Ps, whereas either agentemployed alone did not reduce P2P levels (data not shown). Althoughthese results are preliminary, they suggest that the chemical inductionof terminal differentiation and the irreversible loss of proliferativecapacity in human melanoma cells is associated with a reduction in P2Ps.

B. Gene Expression Changes Induced in Human Melanoma Cells DisplayingReversible Growth Suppression. Reversible Commitment to Differentiationand Terminal Cell Differentiation.

Applicants have begun to determine the spectrum of gene expressionchanges associated with growth suppression, morphologic alterations,increased melanin synthesis, enhanced tyrosinase activity and/orinduction of terminal differentiation in human melanoma cells (12-14).The agents applicants have chosen result in reversible growthsuppression, induction of melanin synthesis, morphologic alterations,enhanced tyrosinase activity, induction of a reversible commitment todifferentiation or terminal differentiation with a concomitant loss ofproliferative capacity in HO-1 melanoma cells (Table 2). The genesapplicants have currently analyzed include: early response genes (c-fos,c-myc, c-jun, jun-B, jun-D and gro/MGSA) (14); interferon stimulatedgenes (ISG-15, ISG-54, HLA Class I and HLA Class II) (13, 14); celladhesion molecules (P-cadherin, E-cadherin, N-cadherin and N-CAM) (14);extracellular matrix genes (fibronectin (FIB) and tenascin) (12, 14);cell surface proteoglycans/matrix receptors (syndecan, β₁ integrin(major FIB receptor subunit), α₅ integrin (major FIB receptor subunit))(14); cytoskeleton genes (tropomyosin-1, γ-actin and β-actin) (14); anda housekeeping gene (GAPDH) (14). Using the gene probes indicated above,no unique gene expression change was found which only occurred interminally differentiated HO-1 cells. These results indicate thatcommitment to differentiation and terminal differentiation in HO-1melanoma cells is associated with specific patterns of overlapping geneexpression changes. As will be discussed below, an interesting change ingene expression observed in both HO-1 cells committed to differentiateand induced to terminally differentiate was the induction and enhancedexpression of type I interferon responsive genes and the gro/MGSA gene.These results have led to the hypothesis that specific autocrinefeedback loops may contribute or are associated with the differentiationprocess in human melanoma cells.

C. Changes in Cell Cycle and Early Immediate Response Genes During theInduction of Terminal Differentiation in Human Melanoma Cells.

As discussed above, treatment of HO-1 cells with the combination ofIFN-β+MEZ results in growth suppression that is apparent by 24 hrfollowing exposure to these inducing agents (FIG. 12) (10, 14). Thissystem has been used to evaluate the effects of the different inducers,alone and in combination, on the expression of cell cycle regulatedgenes, including cdc2, cyclin A, cyclin B, histone 1, histone 4,proliferative cell nuclear antigen (PCNA), c-myc, p53 and Rb. Thesestudies can be summarized as follows: (a) A reduction in cdc2 andhistone 1 was apparent under all treatment conditions. This effect wasobserved after 24 hr and was most dramatic in cells treated for 96 hr;(b) c-myc expression was marginally decreased by 24 hr treatment withMEZ and IFN-β+MEZ, whereas significant suppression was observed by 96 hrespecially in IFN-β+MEZ treated HO-1 cells; (c) Both PCNA and p53 geneexpression was reduced only in cells treated with IFN-β+MEZ; and (d) Rblevels remained unchanged following any of the treatment protocols. Inthe case of cdc2 and histone 1, IFN-β+MEZ resulted in a decreased rateof transcription of these genes. Similarly, IFN-β+MEZ decreased thestability of the cdc2 and histone 1 mRNAs. Analysis of cell cycledistribution by FACS analysis indicated that both MEZ and IFN-β+MEZreduced the number of HO-1 cells undergoing DNA synthesis by 48-hourtreatment. The most effective inhibitor of DNA synthesis in HO-1 cellswas IFN-β+MEZ. These results indicate that the induction of terminaldifferentiation in HO-1 cells by IFN-β+MEZ is associated with asuppression in specific cell cycle related genes that occur at both atranscriptional and a postranscriptional level.

c-fos, c-jun and jun-B expression were superinduced in HO-1 cellstreated with cycloheximide and IFN-β+MEZ, indicating that these genesare immediate early response genes. Differences in the temporal kineticsof induction and the mechanism of enhanced expression were apparentbetween these early response genes in differentiation-inducer treatedHO-1 cells (55). In the case of c-fos, IFN-β+MEZ induced an increase intranscription of c-fos mRNA that was apparent after 1, 6 and 24 hr, butnot after 96 hr treatment (55). In the case of c-jun, increased mRNA wasapparent after 1, 6, 24 and 96-hour treatment with IFN-β+MEZ. Thesechanges in c-jun level did not involve increased transcription, butinstead resulted from an increase in half-life of the c-jun transcripts(55). In the case of jun-B, IFN-β+MEZ increased both the transcriptionand steady-state levels of RNA after 1- and 24-hour treatment. Highlevels of jun-B mRNA were also apparent in HO-1 cells induced toterminally differentiate after treatment with IFN-β+MEZ. The continuedincrease in c-jun and jun-B mRNA levels in terminally differentiatedHO-1 cells suggests that these genes may contribute to maintenance ofthe terminal differentiation phenotype (55).

D. Autocrine Loops Induced in Human Melanoma Cells Treated with IFN-βplus MEZ.

As indicated above, continuous treatment with IFN-β+MEZ for 96-hourresults in terminal differentiation in HO-1 human melanoma cells. Thisprocess correlates with specific patterns of gene expression changes,including the induction of two interferon stimulated genes, ISG-15 andISG-54, and melanocyte growth stimulatory activity (gro/MGSA) (14).These observations suggested the possibility that induction of areversible commitment to differentiation and terminal differentiationmight be associated with the production of differentiation promotingfactors (DPFs) (possibly including IFN-β or an IFN-β-like cytokine andmelanoma growth stimulatory activity (gro/MGSA)). These DPFs could theninduce by an autocrine mechanism the transcription and steady-state mRNAexpression of interferon stimulated genes (ISG) and the gro/MGSA gene inHO-1 cells reversibly committed to differentiation or terminallydifferentiated. To further explore the relationship between treatmenttime and induction of differentiation and to test the autocrinehypothesis, applicants performed two types of experiments. In the firstset of studies (In-Out), HO-1 cells were treated with various agents(including IFN-β+MEZ, MPA+MEZ, PA +MEZ and MEZ (at a high dose of 50ng/ml) for 24 hr, cells were washed 2× with DMEM without FBS, DMEMcontaining 10% FBS was added to cultures and total cytoplasmic RNA wasisolated 72 hours later. For the second set of experiments(Conditioned-Medium), cells were processed as indicated above for In-Outexperiments except after 72 hour growth in the absence of inducer,medium was collected (and contaminating cells were removed bycentrifugation).

Using the In-Out and Conditioned-Medium protocols it was demonstratedthat: (a) At the doses employed, IFN-β+MEZ is a more potent inducer ofgene expression changes and the only combination capable of inducingterminal differentiation in HO-1 cells; and (b) Conditioned medium fromHO-1 cells reversibly committed to differentiate or terminallydifferentiated by IFN-β+MEZ induce specific programs of gene expressionchanges in HO-1 cells that are similar to those induced directly by theinducing agents (14). In addition, conditioned medium from IFN-β+MEZtreated HO-1 cells also induces morphologic changes and suppressesgrowth when added to HO-1 or FO-1 human melanoma cells (data not shown).These results support the hypothesis that induction of terminaldifferentiation in HO-1 melanoma cells is associated with specificchanges in gene expression, some of which may be mediated by orassociated with an autocrine feedback mechanism.

E. Cloning of Melanoma Differentiation Associated (mda) Genes Induced inHO-1 Melanoma Cells treated with IFN-β plus MEZ.

The ability to identify and isolate differentially expressed genesbetween two similar or different cell types is now readily achievableusing subtraction hybridization (rev. 57, 58). A procedure forconstructing subtracted libraries have been developed that is bothsensitive and efficient (49). The application of this approach for theidentification of genes differentially expressed in HO-1 cells treatedwith IFN-β plus MEZ is outlined in FIG. 8. Tester and driver cDNAlibraries are directionally cloned into the commercially available λUni-ZAP phage vector. Subtraction hybridization is then performedbetween double-stranded tester DNA and single-stranded driver DNAprepared by mass excision of -the libraries. The subtracted cDNAs areefficiently cloned into the λ Uni-ZAP phage vector that can be easilymanaged for both screening and gene characterization. The applicabilityof the procedure was demonstrated by the identification of cDNAsdisplaying enhanced expression in human melanoma cells, HO-1, induced toterminally differentiate by treatment with IFN-β+MEZ (FIG. 10). A singleround of subtraction of untreated HO-1 control (Ind⁻) cDNAs fromIFN-β+MEZ treated (Ind⁺) cDNAs generated a series of cDNAs displayingdifferential expression in untreated versus differentiationinducer-treated HO-1 cells, termed melanoma differentiation associated(mda) cDNAs. Employing the approach briefly described above, a total of23 differentially expressed mda cDNAs have been isolated whichrepresents only a portion of the subtracted HO-1 IFN-β+MEZ cDNA library.Partial sequence analysis of these 23 mda genes resulted in theidentification of known genes, including a human TPA-inducible gene, thehuman apoferritin H gene, the IFP-53 (gamma-2 protein) gene, the IL-8(monocyte-derived chemotactic factor) gene, the vimentin gene, the hnRNPA2 protein gene, human macrophage inflammatory protein (GOS19-1) and theIFN-β-inducible gene ISG-56. In addition, 6 cDNAs have been identifiedwhich do not have sequences previously reported in any of the gene databases. As predicted based on the subtraction protocol employed, some ofthe mda genes are induced within 24 hours by: IFN-β and IFN-β+MEZ (e.g.,mda-1 and mda-2); MEZ and IFN-β+MEZ (e.g., mda-3); IFN-β, MEZ andIFN-β+MEZ (e.g., mda-4); and only by IFN-β+MEZ (e.g., mda-5 and mda-6)(FIG. 10). A potentially important group of mda genes is represented bycDNAs displaying significantly enhanced expression in HO-1 cells treatedwith IFN-β+MEZ for 96 hours and displaying terminal celldifferentiation, i.e., mda-5, mda-6, mda-7 and mda-9 (all representingnovel genes) (FIG. 15). Additional mda cDNAs which may prove of value inunderstanding growth control in human melanoma cells have beenidentified which are expressed in both terminally differentiated HO-1cells and HO-1 cells induced to undergo a reversible suppression ingrowth by treatment with IFN-β+IFN-γ, i.e., mda-4, mda-5, mda-7 andmda-8 (FIG. 15). Increased expression of a number of mda genes followingtreatment with IFN-β+MEZ are not restricted to HO-1 cells, sinceincreased mda gene expression is also induced in additional humanmelanomas induced to terminally differentiate by treatment withIFN-β+MEZ (data not shown). The studies described above indicate thefeasibility of using subtraction hybridization to identify genes thatmay directly mediate or represent markers of terminal differentiation inhuman melanoma cells.

F. Gene Expression Changes Induced in the C8161 Melanoma Cells andChromosome 6 Microcell C8161 Hybrids.

Recent studies by Welch et al. (42) indicate that insertion of a normalchromosome 6 (by the microcell chromosome replacement technique) intothe C8161 human melanoma cell line results in a suppression ofmetastatic, but not tumorigenic potential in nude mice. Treatment ofC8161 cells for 4 or 7 days with IFN-β+MEZ (1000 units/ml+10 ng/ml)results in terminal cell differentiation. In contrast, under similarconditions, C8161 cells containing chromosome 6 (Clone 6.1, 6.2 and 6.3)display morphological changes and growth suppression but cells retainproliferative potential, i.e., the combination of agents induces areversible commitment to differentiation as opposed to terminaldifferentiation. A lack of terminal differentiation in 6.1, 6.2 and 6.3cells was demonstrated by removing the test agents and growth in inducerfree medium (data not shown). Analysis of gene expression in parentalC8161 and 6.1, 6.2 and 6.3 cells indicated differences that correlatedwith the presence of a normal chromosome 6. Specific differences in geneexpression after 4 days incubation with IFN-β and MEZ, alone and incombination, include: (a) induction of IL-8 mRNA (which was identifiedas an mda cDNA in HO-1 cells treated with IFN-β+MEZ) in MEZ andIFN-β+MEZ treated C8161 cells, but not in 6.1, 6.2 or 6.3 cells; (b)induction of HLA Class I antigen mRNA by IFN-β, MEZ and IFN-β+MEZ inC8161, but only by IFN-β and IFN-β+MEZ in 6.1, 6.2 and 6.3 cells; and(c) reduced induction of ISG-15 expression in C8161 cells versus 6.1,6.2 and 6.3 cells treated with IFN-β and IFN-β+MEZ. The studies brieflydescribed above indicate that IFN-β+MEZ is more effective in inducingterminal differentiation in the less differentiated metastatic C8161melanoma cells than the more differentiated 6.1, 6.2 and 6.3 cells. Thismodel system should prove useful in determining the role of specific mdagenes in expression of the tumorigenic and metastatic phenotype by humanmelanoma cells.

Design and Methods

A. Specific Aim #1: Determine the pattern and regulation of expressionof the melanoma differentiation associated (mda) genes in melanocytes,nevi, radial growth phase melanoma, vertical growth phase melanoma andmetastatic melanoma cells.

1. Rationale and General Approach:

Applicants have tested the hypothesis that human melanoma cells displayaberrant patterns of differentiation and by appropriate chemicaltreatment they can be induced to undergo an irreversible loss inproliferative capacity without a loss of viability, i.e., terminal celldifferentiation (10,11,14). Using the combination of IFN-β+MEZapplicants have demonstrated that the reprogramming of human melanomacells to terminally differentiate can be achieved in vitro (10,11,14).On the basis of a second hypothesis, i.e., terminal differentiation isassociated with the selective activation of specific programs of geneexpression, applicants have developed and used a modified subtractionhybridization protocol to identify genes displaying enhanced expressionunder conditions resulting in terminal cell differentiation (49). Thesestudies have resulted in the cloning of a series of genes, termedmelanoma differentiation associated (mda) genes, which display suchspecificity. The purpose of the studies to be described below are to:(a) characterize the mda genes with respect to their level of regulationin HO-1 melanoma cells, i.e., transcriptional versus posttranscriptionalmechanisms of induction; (b) determine if the expression of specific mdagenes correlate with a defined stage in melanoma development; (c)continue screening our subtracted IFN-β+MEZ cDNA library to identifyadditional mda genes which display enhanced expression in growtharrested and terminally differentiated human melanoma cells; and (d) useadditional subtraction steps to enrich for genes only expressed at highlevels in HO-1 cells induced to terminally differentiate.

(a) Defining the level of regulation of mda genes in HO-1 cells treatedwith IFN-β+MEZ: Initial studies will focus on the mechanism by whichIFN-β+MEZ increases the expression of cloned mda genes that aresignificantly upregulated (4- to >20-fold) by this combination ofinducing agents in HO-1 cells. The genes to be analyzed will includemda-5, mda-6, mda-7, mda-8 and mda-9 described in Preliminary Studies,which represent novel IFN-β+MEZ-inducible genes not previously reportedin the Gene Bank or the EMBL gene data base. The order of experimentswill include: (1) determining the temporal kinetics of induction of themda genes; (2) determining if the level of induction of specific mdagenes occurs at a transcriptional level; (3) determining if any of themda genes are immediate early (primary) response genes; and (4)determining if differentiation results in an altered stability of themda transcripts.

(i) The screening strategy used to identify the mda genes involvedNorthern hybridization analysis of RNA isolated from HO-1 cells treatedwith IFN-β, MEZ or IFN-β+MEZ for 24 hr (49). Previous studies indicatedthat exposure to IFN-β+MEZ for 24 hr resulted in a number of geneexpression changes also observed in HO-1 cells induced to terminallydifferentiate after 4 days exposure to this combination of agents(Preliminary Studies) (14). mda genes displaying increased expression inHO-1 cells after 24 hr treatment with IFN-β+MEZ were subsequentlyevaluated for enhanced expression after 4 days treatment with theinducers. mda-5, mda-6, mda-7, mda-8 and mda-9 genes displayed enhancedexpression in HO-1 cells treated with IFN-β+MEZ for 24 or 96 hr. Theseresults indicated that increased expression of the mda genes occurredwithin the first 24 hr of treatment and enhanced expression persistedduring terminal cell differentiation. To determine if any of the mdagenes becomes activated after a short exposure to the inducing agents,temporal kinetic studies will be performed. HO-1 cells will be treatedfor short-time periods (15, 30, 45, 60 and 120 min) with IFN-β, MEZ andIFN-β+MEZ, cytoplasmic RNA will be isolated, electrophoresed on 0.6%agarose gels, transferred to nylon filters and sequentially hybridizedwith the various mda genes and lastly with GAPDH (as a control for equalRNA levels under the various experimental conditions) (14). RNA fromcells treated with the inducers will also be isolated every 2 hr over a48 hr period to determine if any cell cycle kinetic changes occur inexpression of the mda genes. An important question that also will beaddressed is whether continued expression of any of the mda genes isrequired for maintenance of the terminal differentiation phenotype. Thiswill be determined by analyzing RNA isolated from HO-1 cells treatedwith IFN-β+MEZ: continuously for 7 days (cells are terminal, but stillviable); for 4 days followed by incubation in growth medium withoutinducers for an additional 10 days (cells remain terminal); and after 10days in cells treated for 24 hr followed by growth in inducer-freemedium (cells regain proliferative potential, i.e., they display areversible commitment to terminal differentiation).

(ii) The studies described above will indicate if any of the mda genesis induced early after exposure to IFN-β+MEZ. To determine if IFN-β+MEZinduce expression of any of the mda genes by increasing their rates oftranscription, nuclear run-on assays will be performed as describedpreviously (55,58,59). Brief Description of Protocol: Nuclei will beisolated from HO-1 cells either untreated (control) or treated for 1, 6and 24 hr with IFN-β (2000 units/ml), MEZ (10 ng/ml) or IFN-β+MEZ (2000units/ml+10 ng/ml). RNA transcripts previously initiated by RNApolymerase II will be allowed to elongate in the presence of [³² P] UTP.Nuclear RNA will be isolated, purified by passing through a G-50sephadex column followed by denaturing with 0.1M NaOH for 5 min on ice(55). Labeled nuclear RNA will be hybridized to nitrocellulose dotfilters containing 2 μg of plasmid DNA containing the mda genes, GAPDHDNA or pBR322 DNA (negative control) which has been denatured by boilingin 0.1M NaOH for 15 min followed by dilution with cold 2M NaCl (55).

(iii) c-fos, c-jun and jun-B are immediate early (primary) responsegenes, i.e., induction is not dependent on new protein synthesis, butrather utilizes existing transcription factors, in IFN-β+MEZ treatedHO-1 cells (55). To determine if any of the mda genes is an immediateearly (primary) response gene, experiments using the protein synthesisinhibitor cycloheximide will be performed (55). Brief Description ofProtocol: Approximately 2×10⁶ HO-1 cells will be untreated or treatedwith IFN-β (2000 units/ml), MEZ (10 ng/ml) or IFN-β+MEZ (2000units/ml+10 ng/ml) for 1 hr in the absence and presence of 50 μg/mlcycloheximide (added 15 min prior to any other additions) (55). TotalRNA will be isolated, electrophoresed in 0.6% agarose, transferred tonitrocellulose filters and hybridized sequentially with the mda genesand lastly with GAPDH (14, 49, 60). By definition, if any of the mdagenes are immediate early response genes they will be induced within 1hour by IFN-β+MEZ in both the absence and presence of cycloheximide.Another indication that the mda genes are primary response genes wouldbe the phenomenon of superinduction, i.e., the massive over-accumulationof immediate early response transcripts which occur when cells aretreated simultaneously with an inducer and protein synthesis inhibitors(55,61,62).

(iv) The ability of IFN-β+MEZ to increase c-jun expression does notinvolve an increase in transcription, but rather results from anincrease in the stability of c-jun mRNA (55). To determine if IFN-β+MEZalters the expression of specific mda genes by a posttranscriptionalmechanism, studies using the transcription inhibitor actinomycin D willbe performed (55). Brief Description of Protocol: Approximately 2×10⁶HO-1 cells will be untreated or treated with IFN-β (2000 units/ml), MEZ(10 ng/ml) and IFN-β+MEZ (2000 units/ml+10 ng/ml) for 24 hr followed byno addition or the addition of actinomycin D (5 μg/ml) for 30 min, 1 hr,2 hr and 3 hr prior to RNA isolation (55). The RNAs will be analyzed byNorthern hybridization and probing with the different mda genes or GAPDH(14,49,55). Radioautograms will be scanned using a densitometer toquantitate cellular RNA levels (55). These studies will indicate ifIFN-β+MEZ can alter the stability, i.e., the half-life, of any of themda gene transcripts.

Summary: These studies will indicate if mda-5, mda-6, mda-7, mda-8 ormda-9 are primary response genes and if their enhanced expression inhuman melanoma cells treated with IFN-β+MEZ results from atranscriptional and/or a posttranscriptional mechanism.

(b) Analysis of mda gene expression during the process of melanomadevelopment: A basic tenet of our terminal differentiation hypothesis isthat the mda genes may represent genes normally expressed or expressedat higher levels in melanocytes and/or in the early stages of melanomadevelopment, i.e., nevi, early radial growth phase (RGP) primarymelanoma and/or early vertical growth phase (VGP) primary melanoma. Ifthis concept is correct, then a prediction would be that specific mdagenes would display reduced expression in late VGP melanoma andmetastatic melanoma versus melanocytes, nevi and early stage melanomas.Support for this hypothesis comes from preliminary studies indicatingthat SV40-transformed human melanocytes express high levels of mda-5,mda-6 and mda-7 mRNA in the absence and in the presence of IFN-β+MEZ(data not shown). To directly test this hypothesis applicants willanalyze RNA obtained from cell cultures of melanocytes, dysplastic nevi(DN 91D, DN (MM92E)), RGP melanoma (WM 35), early VGP melanoma (WM 793,WM902b), advanced VGP melanoma (WM 983a, WM 115) and metastaticmelanomas (WM 9, MeWo, SK-MEL 28, WM 239) (1,16,21,22,27) (to besupplied by Dr. Meenhard Herlyn). Applicants realize that cell culturesmay not always reflect processes occurring in vivo, however, cellcultures will provide an initial indication of which mda genes toemphasize using in situ hybridization approaches with clinical specimensof melanocytes, pre-malignant skin lesions, primary melanoma andmetastatic melanoma. The procedures to be used for in situ hybridizationwith oligonucleotide probes will be as described by Reed et al. (63) andBiroc et al. (64) (to be performed in collaboration with Dr. Anthony P.Albino, Memorial Sloan-Kettering Cancer Center, New York). In thestudies by Reed et al. (63) in situ hybridization with a basicfibroblast growth factor (bFGF) oligonucleotide has been successfullyused to determine differential expression of bFGF in melanocytic lineagetissue specimens. As an additional approach for determining mda geneexpression in clinical specimens, RNA isolated directly from patientsamples displaying different stages of melanoma evolution (to besupplied by Dr. Herlyn) will be evaluated by Northern analysis (14,49)and where necessary to increase sensitivity of detection by RT-PCRanalysis (65) for expression of the mda genes. In previous studiesanalyzing expression of the epidermal growth factor receptor, a widespectrum of human central nervous system tumors obtained from patientswas evaluated (66). This study clearly indicated that intact andhigh-quality RNA could be obtained efficiently from patient material andutilized for comparative gene expression studies.

As indicated in Background and Significance, metastatic melanoma cellsare often inhibited in their growth by TPA (or MEZ), whereas the invitro growth of normal melanocytes and nevi are stimulated by TPA (orMEZ) (1,15,16,20-23). Similarly, many metastatic melanoma cell lines aregrowth inhibited by IFN-β (8,13-15), whereas under optimal growthconditions normal melanocytes are not growth inhibited by IFN-β (eventhough TPA is incorporated in the growth medium) (15). These resultsindicate that progression from melanocyte to malignant melanoma involvesa change in responsiveness to both TPA (or MEZ) and IFN-β. Based onthese observations, it would be predicted that a stage-specific effectwill be observed in melanocyte lineage cells exposed to the combinationof IFN-β+MEZ. If this effect is observed, it will provide a direct testof the potential involvement of the mda genes in the process of growthinhibition and terminal differentiation resulting from treatment withIFN-β+MEZ. The melanocyte lineage cell lines, i.e., melanocyte,dysplastic nevus, RGP primary melanoma, early VGP primary melanoma,advanced VGP melanoma and metastatic melanoma (supplied by Dr. MeenhardHerlyn, Wistar Institute, Philadelphia, Pa.), will be used to directlydetermine: (1) if the combination of IFN-β+MEZ displays a stage-specificgrowth inhibitory effect and the induction of terminal differentiation;and (2) if the induction of growth suppression and/or the induction ofterminal differentiation in stage-specific melanocyte lineage cellscorrelates with changes in the expression of specific mda genes. Thesestudies will be conducted as described previously (10,14). BriefDescription of Protocols: For growth studies: cells will be seeded at2.5 or 5×10⁴ cells/35 mm plate; 24 hr later the medium will be changedwith no additions (Control), 1000 and 2000 units/ml of IFN-β, 1, 10 and50 ng/ml MEZ and combinations of IFN-β+MEZ; cell numbers will bedetermined daily (with a medium change at day 4) over a seven dayperiod. For reversibility studies, cells will be treated for 24 hr or 4days with the different inducers followed by growth in inducer freemedium for an additional 7 to 14 days at which time cell numbers will bedetermined. RNA will be isolated from cells treated for 24 hr, 4 daysand 7 days and analyzed by Northern blotting hybridization forexpression of the mda genes as described previously (14). Biochemicalmarkers for growth suppression and differentiation will include: ananalysis of P2P levels using appropriate monoclonal antibodies andWestern blotting analysis (Preliminary Studies) (52,53); antigenicmarkers, such as the GD3 ganglioside and Class II MHC, and fluorescenceactivated cell sorter analysis as described previously (50,67,68); and adetermination of melanin levels as described previously (69).

Studies designed to identify stage-specific effects of IFN-β+MEZ and therole of the mda genes in growth suppression and terminal celldifferentiation in melanocyte lineage cells will be aided by using threerecently described model systems. These will include: (A) thetransformed human melanocyte cell lines 10Wras/early and 10Wras/late(supplied by Dr. Anthony P. Albino, Memorial-Sloan Kettering CancerCenter, NY, N.Y.) (20); (B) the metastatic human melanoma cell lineC8161 and the tumorigenic but non-metastatic C8161 clones containing anormal human chromosome 6 (supplied by Dr. Dan Welch, Milton S. HersheyMedical Center, Hershey, Pa.) (42,43); and (C) RGP or early VGP primaryhuman melanomas (WM 35, WM 1341B and WM 793) which have been selected byinjection with matrigel in nude mice for a more progressed tumorigenicand metastatic phenotype (e.g., 35-P1-N1, 35-P1-N2, 35-P1-N3,1341-P1-N1, 1341-P1-N2, 1341-P2-N1 etc. (cell line: passage number; nudemouse number)) (supplied by Dr. Robert S. Kerbel, Sunnybrook MedicalCenter, Toronto, Canada) (25,70). The 10Wras/early and 10Wras/late cellsare human melanocytes transformed by a retrovirus containing the viralHa-ras oncogene which display specific traits associated with melanomaprogression (20). 10Wras/early cells display TPA dependence, arenontumorigenic in nude mice, and express many of the antigenic markerspresent in normal melanocytes (20). In contrast, the 10Wras/late cellsare inhibited by TPA, tumorigenic in nude mice, contain many of thecytogenetic changes seen in metastatic melanoma (including modificationsin chromosome 1, 6 and 9) and express many of the same growth factorgenes as metastatic human melanoma (20). As discussed in PreliminaryStudies, applicants have begun to analyze gene expression changes inIFN-β+MEZ treated C8161 cells and C8161 cells containing amicrocell-transferred normal chromosome 6 (6.1, 6.2 and 6.3). UnlikeC8161 cells, 6.1, 6.2 and 6.3 cells are not metastatic in nude mice (42)and they are not induced to terminally differentiate when treated withthe same concentration of IFN-β+MEZ resulting in terminaldifferentiation in C8161 cells. 6.1, 6.2 and 6.3, therefore, mayrepresent human melanoma cells which have been reverted to a lessadvanced stage in melanoma development. As discussed previously, sincesurgical removal of RGP or early stage VGP primary human melanomasresults in a cure of this disease, it has not previously been possibleto analyze more aggressive variants derived from the same early stagemelanomas. Dr. Kerbel and colleagues (25) have potentially overcome thisproblem by injecting RGP and early VGP primary human melanomas incombination with matrigel into nude mice. Tumors which then developedwere found to be tumorigenic in nude mice without the requirement formatrigel and they also acquired a "cytokine resistance phenotype" whichis associated with melanoma progression (25,70). These three cellsystems should prove extremely valuable in determining if mda geneexpression correlates with specific stages of melanoma progression. Totest this possibility applicants will conduct similar experiments asdescribed previously (4. A. 1. a) using these stage-specific cell lines.If our hypothesis suggesting that the response of melanocyte lineagecells to IFN-β+MEZ is stage-specific is correct, then applicants wouldpredict that the effect of these agents on growth, mda gene expressionand terminal cell differentiation would be greater in 10Wras/late vs.10Wras/early cells, C8161 cells vs. chromosome 6 containing C8161 clonesand the more progressed vs. the less progressed RGP and. early VGPprimary melanoma cell lines.

Summary: These studies will indicate if a direct relationship existsbetween the state of progression of melanoma cells and mda geneexpression. They will also indicate if the response to IFN-β+MEZ inducedgrowth suppression, mda gene expression and terminal celldifferentiation is directly related to melanoma progression.

(c) and (d) Identification of additional mda genes which displayenhanced expression in growth arrested and/or terminally differentiatedhuman melanoma cells: As indicated in Preliminary Studies (E), only asmall percentage (approximately 2.5t) of our subtracted IFN-β+MEZ HO-1library has been screened for differentially expressed genes associatedwith growth suppression and terminal differentiation in HO-1 cells. Itis therefore conceivable that a number of additional mda genes, whichstill remain to be identified, are present in the subtracted IFN-β+MEZHO-1 library. The initial subtraction hybridization approach applicantshave used has resulted in the identification of cDNAs displayingenhanced expression in HO-1 cells treated with: IFN-β and IFN-β+MEZ; MEZand IFN-β+MEZ; IFN-β, MEZ and IFN-β+MEZ; and only IFN-β+MEZ (49). Toidentify additional mda genes which are preferentially expressed atelevated levels in cells induced to terminally differentiate followingtreatment with IFN-β+MEZ applicants will perform additional subtractionhybridization steps. Brief Description of Protocol: cDNA libraries willbe constructed from HO-1 cells treated with IFN-β (2000 units/ml) or MEZ(10 ng/ml) for 2, 4, 8, 12 and 24 hr as previously described (49,57).The IFN-β and MEZ cDNA libraries will be converted into single-strandedDNA which will then be biotinylated using photoactivatable biotin andused as the Driver DNA as described previously (49). The HO-1 IFN-β+MEZ(Ind⁺) subtracted cDNA library will be converted into double strandedDNA and the double-stranded inserts will be isolated and used as theTester DNA (49). The Driver DNA will then be subtracted away from theTester DNA resulting in an enriched HO-1 IFN-β+MEZ (Enriched-Ind⁺)subtracted cDNA library (49). As an alternate approach to specificallyidentify genes displaying increased elevation in terminallydifferentiated melanoma cells, cDNA libraries will be constructed fromHO-1 cells treated for 4 or 7 days with IFN-β, MEZ and IFN-β+MEZ.Subtraction hybridization will then be conducted as described (49) usingthese libraries to identify cDNAs only expressed at increased levels inIFN-β+MEZ-treated terminally differentiated HO-1 cells.

Summary: Additional screening of our current HO-1 IFN-β+MEZ (Ind⁺)offers the potential of identifying more differentially expressed mdagenes. By constructing additional cDNA libraries from HO-1 cells treatedsingularly with IFN-β or MEZ and subtracting this information away fromcDNA libraries prepared from HO-1 cells treated with IFN-β+MEZ, theidentification of additional mda genes displaying enhanced expressionspecifically in terminally differentiated melanoma cells should result.

B. Specific Aim #2: Analyze the relationship between mda gene expressionand the induction of reversible commitment to differentiation, growthsuppression without the induction of differentiation, DNA damage andstress responses and induction of terminal differentiation in humanmelanoma and other model differentiation systems.

1. Rationale and General Approach:

Induction of terminal differentiation in human melanoma cells, as wellas other cell types such as myoblasts, neuroblastoma and leukemic cells,is associated with an irreversible loss in proliferative ability (rev13,71). It is therefore reasonable to assume that some of the mda genesapplicants have identified may also display enhanced expression ingrowth arrested melanoma (and other cell types) or melanoma cells (andother cell types) treated with various DNA damaging and chemotherapeuticagents which also induce growth-related changes. Indeed, preliminarystudies indicate that mda-4, mda-5 and mda-8 exhibit increasedexpression in terminally differentiated HO-1 cells as well as HO-1 cellsinduced to undergo reversible growth suppression by treatment withIFN-β+IFN-γ (50). In addition, mda-4 also displays increased expressionin HO-1 cells reversibly growth suppressed by caffeic acid phenethylester (72), vinblastine, tumor necrosis factor-α and X-irradiation.Additional mda genes have been identified which display enhancedexpression only in HO-1 and other metastatic human melanoma cellstreated with IFN-β+MEZ (i.e., they appear to be melanoma specific) or inHO-1 and dissimilar cell types induced to lose proliferative capacity,including human breast and colon carcinoma (i.e., they appear to begrowth and or differentiation specific and not melanoma specific). Basedon these preliminary observations, it appears that specific mda genesmay be restricted to melanoma lineage cells induced to lose growthpotential and become terminally differentiated, while other mda genesmay represent key genes involved in growth control processes in diversecell types. It will, therefore, be important to determine whetherchanges in the expression of specific mda cDNAs are restricted to humanmelanoma cells induced to terminally differentiate or whether they alsodisplay modified expression during other programs of terminaldifferentiation, DNA damage and growth arrest. The studies describedbelow are designed to determine: (a) the spectrum of cellular changeswhich induce enhanced mda gene expression in human melanoma and othercell types; and (b) if induction of growth suppression and terminaldifferentiation in other cell types results in the enhanced expressionof specific mda genes.

(a) Analysis of mda gene expression in human melanoma (and other celltypes) treated with growth suppressing agents, DNA damaging agents andchemotherapeutic agents: Since terminal differentiation in HO-1 cells isassociated with an irreversible loss in proliferative capacity(10,11,14), the mda genes applicants have identified may represent genesinvolved in both cell growth and terminal cell differentiation. Toexplore the relationship between cell growth and terminaldifferentiation, studies will be conducted to determine the types ofagents and treatment protocols which can induce mda gene expression inhuman melanoma and other human cell types. The agents and treatments tobe tested will include: growth suppression (incubation in reduced serumlevels), heat shock, gamma irradiation, ultraviolet irradiation (UVA andUVB), carcinogenic and mutagenic agents (methyl methanesulfonate, ethylmethanesulfonate and 4-nitroquinoline-oxide), demethylating agents(5-azacytidine, phenyl butyrate), chemotherapeutic agents (vinblastine,vincristine, adriamycin, cis-platinum), tumor necrosis factor-α, proteinsynthesis inhibitors (cycloheximide, puromycin, anisomycin), DNAsynthesis inhibitors (amphidicolin, hydroxylurea, ara-C), transcriptioninhibitors (actinomycin D), topoisomerase inhibitors (camptothecin),poly-ADP-ribose inhibitors (3-aminobenzamide), protein kinase Cactivators (TPA, teleocidin, synthetic PKC activators (ADMB and DHI)(73-75)) and phosphatase inhibitors (calyculin, okadaic acid). Initialstudies will focus on HO-1 cells. Subsequent investigations will involveother melanoma cells (representing different stages of melanomaprogression) and additional human cell types (normal fibroblast andepithelial cells, neuroblastoma, glioblastoma, carcinomas (prostate,breast and colon) and sarcomas). Brief Description of Protocol: HO-1cells (or the other experimental cell type employed) will be treatedwith the various agents for different time periods (ranging from 1 hr to24 hr; or with certain treatment protocols for 4 and 7 days) and withdifferent doses of the test treatment or agent. RNA will be isolated,electrophoresed in 0.6% agarose gels, transferred to nylon filters andhybridized sequentially with the different mda genes and lastly withGAPDH. If specific pathways appear to induce an mda gene then furtherbiochemical studies will be conducted. For example, if PKC activatorsinduce specific mda genes then studies will be performed using specificinactive analogs and inhibitors of PKC to determine the relationshipbetween PKC activation and induction of gene expression. Similarly, if atreatment protocol or agent is found to induce or enhance expression ofan mda gene, then subsequent studies will be conducted to determine ifthis change in gene expression is transcriptional orpost-transcriptional (see Specific Aim #1 for experimental details).

Summary: The studies briefly outlined above will indicate if mda geneexpression can be induced in HO-1, other human melanoma cells andadditional human cell types, by treatment with agents which can altergrowth and/or differentiation. They will provide initial informationrelative to potential biochemical pathways which may mediate theinduction or enhanced expression of the mda genes. These experimentswill also identify which mda gene(s) to use in studies (described in C.Specific Aim #3.) designed to determine the potential functionalsignificance of mda gene expression changes in the control of growth anddifferentiation in human melanoma cells and other human cell types.

(b) Analysis of mda gene expression during the process of terminaldifferentiation in human promyelocytic leukemia (HL-60) and humanskeletal muscle cultures: Specific mda genes are expressed in humanmelanoma and additional human cell types undergoing growth suppressionwith and without the induction of terminal differentiation. To explorethis phenomenon further and to determine if any of the mda genes arealso expressed at elevated levels in additional differentiation modelsystems applicants will conduct experiments using the HL-60promyelocytic leukemic cell line (76) and human skeletal muscle cells(77,78). TPA induces macrophage differentiation in HL-60 cells (79),whereas dimethyl sulfoxide (DMSO) results in granulocyticdifferentiation in HL-60 cells (80). In addition, growth of HL-60 cellsin medium containing DMSO for 5 days followed by growth in TPA resultsin cells which switch from a granulocytic to monocytic differentiationprogram (81). These studies indicate that specific monocytic orgranulocytic lineages can be induced in HL-60 cells by appropriatechemical manipulation. By growing HL-60 cells in incremental increasesof TPA and DMSO, applicants have isolated variant populations whichdisplay a quantitative resistance to either TPA- or DMSO-induceddifferentiation (76). These resistant variants do not, however, displaycross-resistance to other inducers, i.e., TPA induces a similar patternof differentiation in parental HL-60 and HL-60/DMSO^(R) cells and DMSOinduces a similar pattern of differentiation in parental HL-60 andHL-60/TPA^(R) cells. Although IFN-aA and IFN-β induce growth suppressionin parental HL-60, TPA-resistant HL-60 (HL-60/TPA^(R)) andDMSO-resistant HL-60 (HL-60/DMSO^(R)) cells, they do not induce thesecells to differentiate terminally (76). However, the combination ofIFN-αA or IFN-β and TPA results in a synergistic growth suppression andthe induction of terminal differentiation in both parental andHL-60/TPA^(R) cells (76). Similarly, the combination of IFN-αA or IFN-βand DMSO results in synergistic growth suppression and the induction ofterminal differentiation in both parental and HL-60/DMSO^(R) cells (76).This experimental model will prove extremely valuable in determining ifa correlation exists between enhanced mda gene expression and eithergrowth suppression or growth suppression with the induction of specificprograms of terminal differentiation in human myeloblastic leukemiccells. Brief Description of Protocols: Parental HL-60, HL-60/TPA^(R) andHL-60/DMSO^(R) cells will be incubated with IFN-β (2000units/ml)±inducer (TPA at 10⁻⁹ and 10⁻⁶ M or DMSO 0.9 to 1.5%) for 1, 3and 7 days (with fresh medium±additions added at day 4). Cell numbersand terminal differentiation (as monitored by the presence ofmorphologically mature cells and the ability of cells to reducenitroblue tetrazolium (NBT) (granulocyte specific) or the production ofnonspecific esterase (macrophage specific) will be determined (84). RNAwill be isolated, electrophoresed in 0.6% agarose gels, transferred tonylon filters and hybridized sequentially with the different mda genesand lastly with GAPDH (14, 49). Since the induction of both growthsuppression and terminal differentiation is concentration- andcompound-dependent in parental HL-60 and variant HL-60 cells, thestudies outlined above will indicate if a relationship exists betweenthe degree of growth suppression and/or the induction of terminaldifferentiation and expression of the different mda genes in HL-60cells.

Methods are available for the in vitro growth of myogenic musclesatellite cells obtained from normal adult human skeletal muscle (82).These cultures recapitulate normal myogenesis, a process that can befollowed with specific morphologic and biochemical markers and thusprovides a useful system for assessing the effects of various agents onthe process of cellular differentiation in human cells (77, 78, 82).Using this model system, applicants have previously demonstrated thatTPA (and related compounds) inhibit spontaneous and induced myogenesis,whereas IFN-αA enhances myogenesis (77, 78). Inhibition or enhancementof differentiation in human skeletal muscle cultures is associated witheither the suppression or induction, respectively, of specificmorphologic changes (development of multinucleated myotubes) and changesin creatine kinase isoenzyme transition from CK-BB to the CK-MM form(77,78). Applicants have also developed SV40-immortalized human skeletalmuscle cells which fail to undergo terminal differentiation when treatedwith IFN-αA (83). This experimental model will prove extremely valuablein determining if any of the mda genes are differentially expressedduring the induction of terminal differentiation or growth suppressionin human skeletal muscle cells. Brief Description of Protocols: Musclecultures will be grown from human skeletal muscle biopsy specimensobtained from diagnostic evaluation (from patients of either sex andranging from 6 months to 50 years of age) as previously described(77,78,82). Prior to myoblast fusion, the cells will be trypsinized andplated at 2×10⁶ cells/10 cm tissue culture plate, cultures will beincubated with IFN-β (2000 units/ml) and MEZ (10 ng/ml), alone and incombination, for 1, 4 and 7 days (with an appropriate medium change atday 4). Cell numbers will be determined and differentiation will bemonitored using morphologic (myoblast fusion) and biochemical (creatinekinase isoenzyme transition from CK-BB to CK-MM) criteria (77,78,82).RNA will be isolated, electrophoresed in 0.6% agarose gels, transferredto nylon filters and hybridized sequentially with the different mdagenes and lastly with GAPDH. When only small quantities of RNA areavailable, RT-PCR (65) will be used to determine expression of theappropriate mda gene in early passage human skeletal myoblast cultures.In this model system, IFN-β promotes skeletal muscle differentiationwhereas MEZ inhibits differentiation. By employing specificconcentrations of IFN-β+MEZ, it is possible to obtain either anenhancement in differentiation, no change in differentiation or aninhibition in differentiation (78). This system should permit a directdetermination of the relationship between mda gene expression and theinduction of myogenesis in human skeletal muscle cultures.

Summary: The studies described briefly above will indicate if any ofapplicants mda genes display altered expression during the induction ofgrowth suppression and terminal differentiation in human myeloidleukemic cells and human skeletal muscle cells. If changes are observed,subsequent studies could be conducted to determine if the mda genesdisplay enhanced expression in other differentiation models, includingthe U937 human monoblastic leukemia cell which can be induced to undergomacrophage differentiation (81), the PC12 rat pheochromocytoma cell linewhich can be induced to undergo neuronal differentiation (85) and humanneuroblastoma cells which can be induced to differentiate terminallywhen treated with retinoic acid or other agents. In addition, if mdagene expression changes are observed in HL-60 cells induced toterminally differentiate, further studies would be conducted using HL-60cells and other inducing agents which result in growth arrest withoutdifferentiation, DNA damage and apoptosis and terminal celldifferentiation (with and without apoptosis). These studies wouldindicate which cellular and biochemical changes in HL-60 cells result ininduction of specific programs of mda gene expression.

C. Specific Aim #3: Isolate full-length cDNAs of mda genes that may beinvolved in melanoma differentiation or progression and directlydetermine their potential functional role in the differentiation andprogression of human melanoma.

1. Rationale and General Approach:

The ability to analyze the functional significance of specific mda geneswill require the isolation of full-length cDNAs. Once a full-length cDNAhas been identified for a specific mda gene it can be used to: (1)produce its encoded protein using an in vitro translation system; (2)generate polyclonal antibodies specific for peptide regions of theencoded protein; (3) determine the location of the mda gene product inhuman melanoma cells and in tissue sections from patients; (4) determinethe effect of overexpression of the mda gene on induction of growthsuppression and terminal differentiation; and (5) determine the effectof blocking mda gene expression (using antisense oligomers or expressionvector constructs) on the ability of IFN-β+MEZ to induce growthsuppression and terminal cell differentiation. The approaches to beutilized to identify full-length mda cDNAs, in vitro translate thefull-length mda cDNAs, produce antibodies against specific peptides ofthe encoded proteins, determine the location of the encoded proteins andto construct and analyze the effect of sense and antisense oligomers andexpression vector constructs on growth and differentiation in HO-1 andother cell types is described below.

(a) Strategy for isolating full-length mda cDNAs: Rapid amplification ofcDNA ends (RACE) is a procedure for amplification of nucleic acidsequences from a mRNA template between a defined internal site and anunknown sequence representing either the 3' or 5' end of the mRNA(86-88). The RACE procedure will be used to obtain the complete sequenceof the full-length mda cDNAs (5' ends) using the sequences (alreadydetermined) as the templates. Two types of gene-specific primers will besynthesized: the RT primer for reverse transcription and the AMP primerfor PCR amplification. The sequence of the AMP primer is locatedupstream of the RT primer. First strand cDNA synthesis is initiated fromthe -RT primer. After first strand cDNA synthesis, the original mRNAtemplate is destroyed with RNase H and unincorporated dNTPs and RTprimers are separated from cDNA using Centricon spin filters (AmiconCorp.). An oligo-DA anchor sequence is then added to the 3' end of thecDNA using TdT (terminal d transferase) and DATP. PCR amplification isaccomplished using the AMP primer and a mixture of oligo(dT)-adapterprimer/adapter primer (1:9). The adapter primer contains a specificsequence which, in the form of dsDNA, can be recognized and digested bythe restriction enzymes SalI and XhoI. Following amplification, the RACEproducts will be digested with specific restriction enzymes (which donot cut the cDNA internally) and cloned into an appropriate plasmid(such as pBlueScript). The clones with specific inserts will be selectedby screening (using DNA filter hybridization) and multiple independentclones of each gene will be used simultaneously for DNA sequencing toeliminate possible errors in sequence determination as a result ofmisincorporations occurring during the PCR amplification process.

(b) Characterization of full-length mda cDNAs: Full-length mda cDNAswill be used to obtain information about the mda-encoded proteins andthe potential function of these genetic elements in regulating growthand differentiation of human melanoma cells. As will be described below,in vitro translation will be used to obtain the mda-encoded proteins.Once the protein structure is determined, synthetic peptides will beconstructed and used to generate antibodies specific for defined regionsof the mda proteins. Antibodies will be used to determine. the locationof the mda proteins in melanoma and other cell types and tissuesections.

(i) Determination of protein structure and development of polyclonalantibodies specific for mda genes: Once full-length mda cDNAs have beenisolated and sequenced, information will be available relative to thepresence of open reading frames and the amino acid composition of theputative proteins encoded by these mda genes. To directly determine thesize of the proteins encoded by the mda genes, full-length cDNAs will besubcloned into the pGEM-1 vector and transcribed in vitro using SP6polymerase (Promega Corp., Madison, Wis.) as described (89). The invitro transcribed RNA will be translated in a rabbit reticulocyte lysate(Amersham) in the presence of [³⁵ S]methionine according to themanufacturer's instructions and dialyzed against 10 mM Hepes, pH 7.9, 1mM MgCl₂, 1 mM dithiothreitol, 20% glycerol, 100 mM NaCl, 100 μM ZnCl₂at 4° C. overnight. Protein products will be analyzed by electrophoresisin an SDS/10-20% polyacrylamide gradient gel. Based on predicted aminoacid structure of the protein, i.e., hydrophobicity value, antigenicityvalue and turn structure based on the deduced sequences of the mda cDNA,specific amino acids will be chosen for generating synthetic peptides(75,90). Synthetic peptides will then be used to generate polyclonalantibodies (Hazelton Laboratories, Denver, Pa. or Cappel Laboratories,Durham, N.C.). Brief Description of Protocol: The synthetic peptideswill be conjugated with carrier proteins, bovine serum albumin and CGG(chicken γ-globulin) as described (90). Two and one-half mg of peptideand 5 mg of carrier proteln in double-distilled water will be incubatedwith 20 mg of either ethyl CDI (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride) (Sigma) or morpho CDI(1-cyclohexyl-3-(2-morpholinoethyl)carbodiimidemetho-p-toluenesulfonate) (Sigma) in water at room temperature for 2hours and dialyzed against PBS, pH 7.2. Rabbits will be immunized with0.5 mg of BSA peptide (conjugated by ethyl CDI) emulsified with completeFreund's adjuvant at 2-week intervals. The antisera against peptideswill then be purified by affinity chromatography coupled with CGGpeptide (conjugated by morpho CDI), excluding the by-product generatedin the conjugation reaction. The polyclonal antibodies will be titered(by 1:1 serial dilution, beginning with a 1:50 dilution) on humanmelanoma cells, either untreated or treated with IFN-β+MEZ, by ELISAassays as described previously (91).

(ii) Immunostaining of cultured cells and sectioned patient samples withanti-mda peptide antibodies: Based on preliminary studies usingSV40-immortalized human melanocytes, applicants would predict thatspecific mda gene products would be produced at increased levels innormal melanocytes versus melanoma cells. To test this possibility andto determine if differences are apparent in specific stages of melanomadevelopment or as a consequence of IFN-β+MEZ treatment, melanocytes,dysplastic nevi and different staged melanomas (RGP, early VGP, late VGPand metastatic) will be cultured on coverslips. Twenty four hr later,one group of cultures will receive a media change without additions andthe other will receive fresh medium with 2000 units/ml of IFN-β+10 ng/mlof MEZ. After an additional 24 hr, cultures will be washed 3× with PBSfollowed by fixation with 3.7% formalin for 30 min (90,91). The slideswill then be washed 3× with PBS and treated with 0.2% Triton X-100/PBSfor 5 min at room temperature. After extensive washing with PBS andblocking nonspecific binding sites with 2% egg albumin/PBS, the cellswill be incubated with affinity purified anti-mda peptide antibody orpreimmunized control sera for 30 min. The slides will then be incubatedwith fluorescein isothiocyanate-conjugated goat anti-rabbitimmunoglobulin antibody for 30 min followed by examination with afluorescence microscope after extensive washing with 0.1% SDS/PBS (90).Studies will also be conducted to determine if the anti-mda peptideantibodies react with melanocyte/melanoma lineage tissue. Reactivity ofthe anti-mda peptide antibodies toward sectioned clinical samplesrepresenting normal melanocytes, dysplastic nevi, and RGP, early VGP,late VGP and metastatic melanoma (supplied by Drs. Albino and Herlyn)(63) will be analyzed as previously described (90,91).

(iii) Analysis of the effect of forced mda expression on growth anddifferentiation in human melanoma cells: A key question will be whetherexpression of any of the mda genes can directly inhibit melanoma growthor induce morphological, biochemical or gene expression changesassociated with growth suppression and the induction of differentiationwithout the addition of inducer (IFN-β+MEZ). To determine the effect ofoverexpression of specific mda genes on growth and differentiation inhuman melanoma cells applicants will employ expression vectorscontaining full-length mda cDNAs. Full-length mda cDNAs will be clonedinto an expression vector containing an inducible promoter, such as thedexamethasone (DEX)-inducible MMTV promoter, and a selectable antibioticresistance gene, such as the neomycin resistance gene, e.g., pMAMneoconstruct (Clonetech). Transfer of this mda-S construct into humanmelanoma cells will permit the direct isolation of cells containing thespecific mda-S gene and will permit regulation (by altering DEXconcentrations) of the level of expression of the specific mda gene.This approach may be necessary, since continuous increased expression ofspecific mda genes under control of promoters such as thecytomegalovirus (CMV) or the β-actin promoter may result in loss ofproliferative ability and/or terminal differentiation in human melanomacells. By using different MMTV promoter-driven constructs containingdifferent mda genes and different antibiotic resistance genes, it willalso be possible to construct melanoma cells containing several mda-Sinducible gene constructs. An additional advantage of a regulatableexpression vector system will be the stable nature of the cell clonesand the ability to determine if transient or prolonged mda-S expressionis required to induce an irreversible loss of proliferative capacity andterminal differentiation in HO-1 cells. Brief Description of Protocols:HO-1 cells (additional human melanoma cell lines or other human tumorcell lines) will be seeded at 2×10⁶ cells/100 mm plate, 24 hr latercells will be transfected by electroporation with the mda-S construct(alone if a selectable gene is present in the construct or inconjunction with a cloned selectable gene) as described (92). Antibioticresistant colonies will be selected and isolated as pure clones (92,93). The presence of the inserted gene will be determined by Southernblotting and expression of the endogenous and exogenous gene will bedistinguished by RNase protection assays (58,94). The ability of DEX toenhance expression of the mda-S gene in appropriate HO-1 cell lines willbe determined by growing cells in the presence or absence of theappropriate inducer (10⁻⁹ to 10⁻⁵ M DEX or 2000 units/ml of IFN-β) for24 hr prior to isolating and characterizing RNA expression (95). Underconditions of non-induction (absence of DEX) or induction (presence ofDEX) cells containing the MMTV-inducible mda-S constructs will beevaluated for alterations in growth (10, 14), increases in melaninsynthesis (10, 69), modification in cell surface antigenic phenotype(67, 68, 72), changes in the levels of P2Ps (52, 53), patterns of geneexpression (14) and the induction of irreversible loss of proliferation(terminal differentiation) (10,11,14) using previously describedprotocols.

Potential Outcome of mda-S Expression Construct Studies: The studiesbriefly described above could result in one of three potential outcomes.First, a single mda-S construct could induce growth suppression, geneexpression changes associated with differentiation and terminal celldifferentiation in the absence of IFN-β+MEZ. This would providecompelling evidence that this specific mda gene is a controlling elementin regulating growth and differentiation in human melanoma cells.Second, specific mda-S constructs could modify only a portion of thechanges induced by IFN-β+MEZ in human melanoma cells, i.e., inducegrowth suppression, induce growth suppression and some markers ofdifferentiation or induce only some of the gene expression changesassociated with differentiation without affecting growth. If thisoccurs, then it may be possible by using a combination of mda-Sconstructs, which induce different components of the differentiationprogram in human melanoma cells, to induce a loss of proliferativecapacity and terminal differentiation in human melanoma cells. Third,mda-S constructs could display no effects on growth or differentiationprograms in HO-1 cells. This result would of course be the leastinformative outcome. It would suggest that the specific mda-S constructstested are not controlling elements in melanoma differentiation,although these genes may be altered during the induction of growthsuppression and terminal differentiation in human melanoma cells byIFN-β+MEZ.

(iv) Analysis of the effect of antisense oligomers and expression vectorconstructs on growth and differentiation in human melanoma cells:Antisense RNA is an effective approach for interfering with theexpression of specific target genes (96). The antisense transcript has asequence complementary to the target mRNA and can presumably anneal tothe mRNA and disrupt normal processing or translation (96). Mechanism(s)by which antisense constructs inhibit gene functions include: a directinterference in translation by binding to the ribosomal assembly(translation initiation) site and/or coding regions; stimulation of mRNAdegradation by RNase H which specifically cleaves double-stranded RNAhybrids; and blocking translocation of the mRNA from the nucleus intothe cytoplasm (96). Previous studies have demonstrated that antisenseconstructs or oliogodeoxyribonucleotides (oligomers) of specific genes,such as c-myc and Egr-1, can modulate cell growth and/or differentiation(84,97-101). To determine the effect of blocking mda gene expression ongrowth and differentiation in human melanoma cells applicants willconduct experiments using antisense oligomers and expression vectorscontaining antisense constructs.

In the first set of experiments applicants will determine if oligomerscomplementary to specific regions of the mda genes can induce growthsuppression and/or changes in the expression of genes previously shownto be altered in HO-1 cells treated with IFN-β+MEZ, i.e., c-myc, IL-8,FIB, MGSA/gro, ISG-15, c-jun and jun-B (14,55). As indicated above,antisense oligomers have been employed successfully to alter cellphysiology in a number of cell lines and they have been shown to affectthe expression of many genes (rev. 102,103). mda-gene specific oligomerscomplementary to the translation initiation sites or 5'-coding regionswill be synthesized with phosphorothioate modification (phosphorothioateoligodeoxynucleotide) to increase nuclease resistance (96,102,103).Although unmodified oligomers have been utilized in many laboratories,they can be degraded rapidly by nucleases present in serum-supplementedmedium (96,102). Since experiments with HO-1 and the other cell typeswill utilize serum-containing medium, applicants will usephosphorothioate oligodeoxynucleotides as opposed to unmodifiedoligomers. Brief Description of Protocols: For growth studies, HO-1cells (or other test cell lines) will be seeded at 2.5×10⁴ cells/35mmtissue culture plate and 24 hr later fresh medium with IFN-β+MEZ (2000units/ml+10 ng/ml), various concentrations (1 to 200 μM) of the3',5'-phosphorothioate end-capped mda antisense oligomer complementaryto several target sequences of the 5' region of the mda gene (20 basesin length) (mda-AS oligomer) or the combination of IFN-β+MEZ and themda-AS oligomer will be added. As appropriate controls, cultures willalso receive similar concentrations of a 5',3'-phosphorothioateend-capped mda sense oligomer (mda-S oligomer) or the mda-S oligomerplus IFN-β+MEZ (65, 84, 104). Cell numbers will be determined daily overa 7 day period (with medium changes with the appropriate additions every48 hr) to identify the correct mda-AS oligomer to use and theconcentration of mda-AS oligomer required to block the effect ofIFN-β+MEZ on growth inhibition in HO-1 cells. If an appropriate mda-ASoligomer is identified it will be used for subsequent studies todetermine the effect of this mda-AS oligomer on gene expression (14) andbiochemical (10, 52, 53, 69) and immunological changes (67, 68, 72)induced in HO-1 cells treated with IFN-β+MEZ. For gene expressionstudies, HO-1 cells (or other test cell lines) will be seeded at 2.5×10⁶cells/100 mm tissue culture plate and the appropriate concentration ofmda-AS or mda-S oligomer will be added 24 hr later. RNA will be isolatedafter an additional 24 hr or 96 hr (with a medium change after 48 hr),electrophoresed in 0.6% agarose gels, transferred to nylon filters andhybridized sequentially with c-myc, c-jun, jun-B, ISG-15, MGSA/gro,IL-8, FIB and lastly with GAPDH (14). Assays will also be conducted todetermine if mda-AS oligomers can prevent or diminish the biochemical,immunological and/or cellular changes induced in HO-1 cells by thecombination of IFN-β+MEZ. Parameters to be monitored, by previouslydescribed techniques, include morphology (10), melanin synthesis(10,69), antigenic expression (67,68,72) and levels of P2Ps (52,53).Possible Outcome of Studies: The experiments described above couldproduce one of three possible outcomes. First, specific mda-AS oligomerscould inhibit the ability of IFN-β+MEZ to induce growth suppression,gene expression changes associated with differentiation and terminalcell differentiation. Second, specific mda-AS oligomers could modifyonly a portion of the changes induced by IFN-β+MEZ in HO-1 cells, i.e.,reverse growth suppression, reverse both growth suppression and terminaldifferentiation or reverse only some of the gene expression changes.Third, specific mda-AS oligomers could display no effect on the growthsuppression or the differentiation program induced by IFN-β+MEZ.

A positive result using mda-AS oligomers in blocking specific cellularand biochemical changes in HO-1 cells treated with IFN-β+MEZ wouldprovide strong evidence for a relationship between expression of aspecific mda gene and a defined component of the differentiationprocess. However, a negative effect of a specific mda-AS gene couldoccur for many reasons including lack of stability of the antisenseoligomer, inadequate quantity of the antisense oligomer or therequirement for the expression of multiple mda genes in thedifferentiation process. To further explore the effect of mda-AS geneexpression in inhibiting chemical induction of differentiation in humanmelanoma cells experiments will also be conducted using mda-AS cDNAs(mda cDNAs cloned in an antisense orientation) in expression vectorconstructs. Since one question applicants intend to address is therelationship between levels of expression and tissue specific expressionof specific mda-AS cDNAs and cellular phenotype, applicants will useseveral expression vector constructs under the transcriptional controlof different promoters. The constructs to be used should result in highlevel targeted, constitutive or inducible transcriptional control of themda-AS cDNAs. The same mda-AS cDNA will be cloned into an expressionvector containing a promoter which will permit: high levels ofconstitutive expression (β-actin promoter (84,94)); enhanced expressionafter interferon treatment (interferon responsive sequence (IRS)promoter (pTKO-1) (105); inducible expression in the presence ofdexamethasone-(MMTV) inducible promoter (93, 106); or expression inmelanocyte/melanoma lineage cells (tyrosinase promoter (107)). Inaddition, by using different selectable genes, i.e., neomycin,histidinol, hygromycin etc., either present in the expression vectorconstruct or by cotransfection (93) it will be possible to constructhuman melanoma cells which contain several mda-AS cDNAs. In summary, theuse of different promoters will permit a direct evaluation ofconstitutive, inducible and cell-lineage specific (targeted) expressionof the mda-AS cDNA on growth and differentiation in human melanoma andother cell types. If expression of a specific mda-AS cDNA inhibits theability of IFN-β+MEZ to induce growth suppression, changes in geneexpression and terminal differentiation in HO-1 and other human melanomacells, this would provide strong evidence for a direct relationshipbetween expression of this mda cDNA and the growth and differentiationprocess in human melanoma cells. Brief Description of Protocols: HO-1cells (additional human melanoma cell lines or other human tumor celllines) will be seeded at 2×10⁶ cells/100 mm plate, 24 hours later cellswill be transfected by electroporation with the mda-AS construct (aloneif a selectable gene is present in the construct or in conjunction witha cloned selectable gene) as described (92, 93). Antibiotic resistantcolonies will be selected and isolated as pure clones (92, 93). Thepresence of the inserted gene will be determined by Southern blottingand expression of the endogenous and exogenous gene will bedistinguished by RNase protection assays (58, 94). The ability ofinterferon or dexamethasone to enhance antisense in a pTKO-1 (interferoninducible promoter) or a pMAMneo (DEX inducible) construct,respectively, will be determined by growing cells in the presence orabsence of the appropriate inducer (10⁻⁹ to 10⁻⁵ M DEX or 2000 units/mlof IFN-β) for 24 hours prior to isolating and characterizing RNAexpression (95, 105). Similar cellular (growth and morphology),biochemical (melanin and P2P levels), immunological (antigenicexpression) and molecular (gene expression) parameters as used to studymda-S constructs will be used to study mda-AS constructs.

Summary: These studies will provide important information about the mdaencoded gene products and they will indicate if perturbations in theexpression of specific mda genes can directly modify growth or thedifferentiation process in human melanoma cells.

D. Specific Aim #4: Isolate and characterize the promoter region of mdagenes and analyze their regulation in human melanocytes, nevi andmelanoma.

1. Rationale and General Approach

In order to elucidate the mechanism underlying the transcriptionalregulation of the mda genes it will be necessary to analyze the promoterregions of these genes. This will be important for studies aimed atdetermining regulatory control of the mda genes including chemicalinduction, autoregulation, tissue specific regulation and developmentalregulation. Once the appropriate promoters of the mda genes have beenisolated, studies can be conducted to identify relevant trans-regulatoryfactors (nuclear proteins) which bind to specific cis-regulatoryelements and activate or repress the expression of the mda genes. Theexperiments outlined below are designed to: [a] clone the promoterregion of specific mda genes and analyze their function in untreated andIFN-β+MEZ treated melanoma cells; [b] identify cis-regulatory elementsin the promoter region of specific mda genes which are responsible forIFN-β+MEZ induction in human melanoma cells; and [c] identify andcharacterize trans-regulatory elements which activate (or repress)expression of the mda genes.

(a) Cloning the promoter region of the mda genes and testing theirfunction in untreated and IFN-β+MEZ treated human melanoma cells: Toisolate the promoter region of the mda gene a human genomic library willbe constructed by partial digestion of HO-1 human melanoma genomic DNAwith the restriction enzyme Sau3AI and then ligation intodephosphorylated vectors (phage or cosmid vectors) (92). Using the mdacDNA gene to screen the library, clones will be identified which containboth the mda gene and its 5' and 3' regions (92). Since the insert in aphage or cosmid vector is too large to analyze (i.e., 10 to 30 Kb) andthe structure and size of the genomic DNA for the mda genes are notknown, the inserts will be subcloned (to an approximate size of 2 Kb) inorder to identify the potential promoter region (92). Two types ofprobes will be used for subcloning: one containing the coding region ofthe mda gene and the other a synthetic oligonucleotide (a 20 mer)complementary to the sequence located in the 5' non-translated region ofthe mda gene. The genomic DNA containing the mda gene in phage or cosmidvector will be digested with a series of restriction enzymes,electrophoresed on 0.8% agarose gels and transferred to nylon filters(92,108). This Southern blot will then be probed with the coding regionof the mda gene and the synthetic oligonucleotide. This double probingmethod will permit a more effective identification of the promoterregion than utilizing a single probe. Putative promoter inserts ofapproximately 2 Kb in size will be subcloned into various CAT expressionvector constructs (including pSVOCAT, pUVOCAT or pChlorAce) for laterfunctional analysis (92,108,109).

The putative promoter region of the mda genes will be sequenced by theSanger dideoxynucleotide procedure (110). The transcription start site(+1) will be determined by primer extension as described previously(108, 111). Dried total RNA samples of HO-1 melanoma cells with orwithout treatment with IFN-β+MEZ will be resuspended in 20 μl of 10 mMPIPES (pH 6.4)-400 mM NaCl containing 5'-end ³² P-labeledoligonucleotides (a 25 mer, complementary to the 5'-untranslated regionof the mda gene) made by the T4 nucleotide kinase method. After 3 hrincubation at 60° C., 80 μl of 50 mM Tris-HCl (pH8.2)-5 mM MgCl₂ -10 mMdithiothreitol-5 mM deoxyguanosine nucleoside triphosphates-25 μl ofdactinomycin per ml containing 10 U of avian myeloblastosis virusreverse transcriptase will be added and the primer extension reactionwill be allowed to proceed for 1 hr at 42° C. Followingphenol-chloroform extraction and ethanol precipitation, samples will beelectrophoresed on 6% acrylamide-8 M urea sequencing gels (108,111).From the length of the extended products, the transcription initiationsite of the mda gene can be determined.

To functionally analyze the various mda promoters, appropriate pmdaCATconstructs will be transfected into melanoma cells by the CaPO₄ methodor electroporation (Gene Pulser, Bio-Rad) as previously described (93,108, 109). Cell extracts will be prepared 48 hr after transfection bywashing cells 3× with PBS, pelleting manually resuspended cells andlysing cells by three cycles of freeze-thawing. The CAT reaction willthen be performed by adding 55 μl of cell extracts into a reactionmixture consisting of 5 μl of ¹⁴ C-chloramphenicol, 70 μl of 1 MTris-HCl (pH8.0) and 20 μl of 4 mM butyrl CoA (108,109). Afterincubation at 37° C. for 2 hr, the reaction mixture will be extractedwith ethyl acetate or xylene and CAT activity will be determined eitherby scintillation counting or by TLC (108,109). If CAT expression can bedetected in specific pmdaCAT construct transfected human melanoma cellsafter treatment with IFN-β+MEZ, but not in untreated cultures, thiswould indicate that the promoter region of the specific mda genecontains appropriate cis-acting elements responsive to induction byIFN-β+MEZ. If no induction is apparent, further subcloning and screeningof cosmid or phage clones would be performed until an mda promoter ofsufficient length to mediate CAT induction in differentiationinducer-treated human melanoma cells is obtained. A potentially usefulseries of CAT constructs have been developed by United StatesBiochemical (Cleveland, Ohio), referred to as the pChlorAce series. Thebasic plasmid pChlorAce-B does not contain a eucaryotic promoter orenhancer sequences and is therefore dependent on incorporation of afunctional promoter upstream from the CAT gene for expression of CATactivity. The construct, pChlorAce-E, contains an enhancer sequencepermitting a direct test of a functional promoter CAT-junction fortesting mda promoter sequences. The promoter containing plasmid,pChlorAce-P, incorporates an SV40 promoter upstream from the CAT geneallowing the insertion of enhancer elements in both orientationsupstream or downstream from the promoter-CAT transcriptional unit. Itwill be possible by inserting different parts of the mda gene into thepChlorAce-P construct to directly identify the enhancer component of themda gene. The control plasmid, pChlorAce-C contains both the promoterand enhancer and can function as an internal standard for comparingpromoter and enhancer strengths.

An important question will be whether specific mda genes display tissue-and developmental-specific expression. Once specific mda promoters areidentified they can be used to address this issue. mda-CAT constructswill be tested for levels of expression in untreated andIFN-β+MEZ-treated normal human melanocytes, dysplastic nevi and RGP,early VGP, late VGP and metastatic melanomas. Experiments will also beperformed to determine if the mda promoters can function in eitheruntreated or IFN-β+MEZ-treated non-melanocyte/melanoma lineage cells.Positive expression in specific target cells would suggest that theappropriate regulatory proteins are either constitutively present orinducible in these cells.

(b) Identifying cis-elements in the mda promoter responsible forinduction by IFN-β+MEZ in human melanoma cells: Once a functional mdapromoter has been identified studies will be conducted to locatecis-elements responsible for induction of expression by IFN-β+MEZ. Theapproach to be used will involve the construction and evaluation for CATinducible activity of a series of 5'-deletion mutants, 3'-deletionmutants, internal-deletion mutants and linker-scanning mutants of themda promoter regions. The details for constructing the 5'-deletion,3'-deletion and internal-deletion mutants and screening for CAT activityhas been described previously (108,109). Linker-scanning mutants will beconstructed by combining 5'- and 3'-deleted mda promoter sequences afterfilling gap regions with a linker DNA sequence (such as an EcoRI linker)(92). The structure of the various mutants will be determined bysequence analysis (95,110). Since the promoter region for the mda geneis located in front of the CAT reporter gene in the various pmdaCATconstructs, the CAT activity for each construct can be measured usingliquid scintillation and/or TLC (108, 109). This will permit a directcomparison of CAT transcriptional activity of the mutant promoter tothat of the unmodified mda promoter. These studies will result in theidentification of specific cis-regulatory elements responsible forIFN-β+MEZ induction of enhanced mda gene expression in human melanomacells.

(c) Identifying trans-acting nuclear proteins induced by IFN-β+MEZ whichmediate transcriptional enhancing activity of mda genes in humanmelanoma cells: The current view on regulation of eucaryotic geneexpression suggests that trans-acting proteins bind to specific siteswithin cis-elements of a promoter region resulting in transcriptionalactivation (rev in 112,113). Studies employing various mutant mdapromoter CAT constructs will provide information relative tocis-regulatory elements mediating activity of the mda promoters.Experiments will be performed to identify trans-acting factors (nuclearproteins) and determine where these factors interact with cis-regulatoryelements. To achieve this goal, two types of studies will be performed,one involving DNase-I footprinting (methylation interference) assays(108,109) and the second involving gel retardation (gel shift) assays(109,114).

For DNase-I footprinting assays nuclear extracts from human melanomacells, untreated or treated with IFN-β+MEZ, will be prepared asdescribed previously (109,113,115). DNase-I footprinting assays will beperformed as described (108,109). The cis-element (approximately 200 bp)for IFN-β+MEZ induction, identified from the experiments describedabove, will be terminally labeled with ³² P and incubated with crudenuclear extracts from untreated or IFN-β+MEZ treated human melanomacells using the protocols described previously (108,109). The reactionmixture which has been digested with DNase-I enzyme will be terminatedand the digested products will be analyzed on an 8% sequencing gel(108,109). The differential protection between nuclear extracts frominduced and uninduced human melanoma cells will provide relevantinformation concerning the involvement of trans-acting factors inactivation and the location of specific sequences in the cis-regulatoryelements of the mda promoter mediating this activation. If differentialprotection is not detected using this approach, the sensitivity of theprocedure can be improved by using different sized DNA fragments fromthe mda promoter region or by using partially purified nuclear extracts(109).

As a second approach for investigating the interaction betweencis-acting elements in the mda promoter and trans-acting factors inmediating transcriptional control, gel shift assays will be performed asdescribed previously (109,114). For this assay, ³² P-labeledcis-elements will be incubated with nuclear extracts from untreated orIFN-β+MEZ treated human melanoma cells and reaction mixtures will beresolved on 5 or 8% polyacrylamide gels (109,114). Afterautoradiography, the pattern of retarded DNAs on the gel will provideinformation concerning the interaction between trans-acting factors andspecific regions of the cis-elements in the mda promoters. Non-labeledcis-elements (self-competition) will be added as a competitor toduplicate samples to eliminate the possibility of non-specific bindingand to confirm that the interaction is really conferred by thetrans-acting factor. To begin to identify the trans-acting factors,different non-labeled DNAs (for example TATA, CAT, TRE, Sp-I bindingsite, NFkB, CREB, TRE, TBP, etc.) can be used as competitors in the gelshift assay to determine the relationship between the trans-actingfactors and other previously identified transcriptional regulators.

Summary: These studies will result in the identification and cloning ofthe mda promoter region, the identification of cis-regulatory elementsin the mda promoters and the identification of trans-regulatory elementswhich activate (or repress) expression of mda genes.

F. Future Studies: The currently proposed research will result in thecharacterization of specific genes which may be involved in or mediategrowth control, response to chemotherapeutic and DNA damaging agents andterminal differentiation in human melanoma cells. Once appropriate mdagenes are identified they can subsequently be used to directly testtheir functional role in development and melanocyte/melanoma biology.Experiments can also be performed to define the role of these genes innon-melanoma target cells and additional programs of differentiation.Future studies which also are not within the scope of the presentproposal would include: (a) evaluation of the effect of specific mda-Sand mda-AS constructs on the growth (tumorigenic and metastaticpotential) and differentiation of human melanoma (and other tumor celltypes) grown in vivo in nude mice; (b) generation of transgenic micedisplaying both tissue and non-tissue specific overexpression ofindividual or combinations of mda genes to evaluate the effect ofmodified mda expression on development; and (c) homologousrecombination-mediated gene targeting techniques to produce mice withspecific mda null mutations to evaluate the effect of lack of expressionof specific mda genes on tissue and embryo development.

References of the Third Series of Experiments

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Fourth Series of Experiments

mda-1: Novel gene which displays increased expression in IFN-β andIFN-β+MEZ treated HO-1 cells after 24 hours. Decreased expression occursin HO-1 cells treated with IFN-β+MEZ for 96 hours. (HJ 3-13).

    (Seq. ID No. 1)                                                                 TGGACTTGTGTTCTGACTAGAACTCAACATGTTACTAGGCACATGTGTCAT                            - GTCTCAGGTCAGTGCTGTGACAGAATTGATACGAGAGAAATGTCGCTTATG                         - CTATCACTGATCTACACATGTCTGATAGATAGTCAGATACAGATGATGAGG                         - AATCT                                                                       - mda-2:                                                                      -  (Seq. ID No. 2)                                                                   GAATTCAGTGAACTCTTTTCTCATTCTCTTTGTTTTGTGGCACTTCACAAT                    - GTAGAGGAAAAAACCAAATGACCGCACTGTGATGTGAATGGCACCGAAGTC                         - AGATGAGTATCCTGTAGGTCACCTGCAGCCTGGCTTGCCACTTGTCTTAAC                         - TCTGAATATTTCATTTCAAAGGTGCTAAAATCTGAAATCTGCTAGTGTGAA                         - CTTGCTCTACTCTCTGAATGATTCAATCCTATTCATACTATCTTGTAGATA                         - TATCAACTAAAAAAA                                                      

Properties of mda-4

This cDNA is novel (analysis of various gene data bases indicates thatthe sequence of mda-4 is 68.5% homologous to the human interferon gammainduced protein).

Expression in HO-1 Human Melanoma Cells

Increased expression after 24 hour treatment of HO-1 cells withrecombinant human fibroblast interferon (IFN-β) (2,000 units/ml),mezerein (MEZ) (10 ng/ml) and to a greater extend with the combinationof IFN-β+MEZ (2,000 units/ml+10 ng/ml).

Analysis of terminally differentiated HO-1 cells, i.e., HO-1 cellstreated with the combination of IFN-β+NEZ (2,000 units/ml+10 ng/ml) for96 hours, indicate continued increased expression in IFN-β+MEZ treatedHO-1 cells.

Increased expression in HO-1 cells after 96 hour exposure to immuneinterferon (IFN-γ) (2,000 units/ml) and IFN-β+IFN-γ (1,000units/ml+1,000 units/ml) (note: this combination of agents results in asimilar degree of growth suppression in HO-1 cells as does IFN-β+MEZ.However, growth suppression is reversible with the combination ofinterferons, whereas it is irreversible with the combination ofIFN-β+MEZ).

mda-4 represents a novel IFN-γ-inducible gene which is also inducedduring terminal cell differentiation in HO-1 cells. Could prove usefulas a gene marker for immune interferon response and as a gene marker forterminal differentiation in human melanoma cells.

Expression in Additional Human Melanoma Cells

Increased expression of mda-4 results after a 24 hour treatment withIFN-β+MEZ in HO-1, LO-1, SH-1, WM278 and WM239 human melanoma cells.Mda-4 is not expressed or inducible in the melanotic FO-1 human melanomacell or in the C8161 human melanoma cells or C8161/6.3 cells (a C8161human melanoma cell clone containing an inserted normal human chromosome6: These cells are tumorigenic in nude mice, but unlike parental C8161cells, they are non-metastatic).

Mda-4 displays increased expression in additional human melanoma cellsbesides the human melanoma cell from which it was cloned, i.e., HO-1after 24 hour treatment with IFN-β+MEZ.

Expression in Normal Cerebellum, a Central Nervous System Tumor(Glioblastoma Multiforme) (GBM) and Normal Skin Fibroblast Cell Lines

Mda-4 is not expressed de novo in normal cerebellum, GBM or normal skinfibroblasts. However, it is inducible in both normal cerebellum and GBM,but not in normal skin fibroblasts, following a 24 hour treatment withIFN-β+MEZ.

Mda-4 is susceptible to induction by IFN-β+MEZ in human cerebellum andGBM cultures, but not in normal human skin fibroblasts.

Expression in Colorectal Carcinoma (SW613), Endometrial Adenocarcinoma(HTB113) and Prostate Carcinoma (LNCaP)

Mda-4 is not expressed de novo in various carcinoma cells and it is notinducible in these cells following a 24 hour treatment with IFN-β+MEZ.Mda-4 is not expressed in human carcinoma cells.

Effect of Various Treatment Protocols on Expression in HO-1 Cells

A 24 hour treatment with IFN-β (2,000 units/ml), IFN-α (2,000 units/ml),IFN-β+MEZ (2,000 units/ml+10 ng/ml), IFN-α+MEZ (2,000 units/ml+10ng/ml), cis-platinum (0.1 μg/ml), gamma irradiation (treated with 3 grayand analyzed after 24 hours). In addition, treatment with UV (10joules/mm² and assayed 24 hours later) results in increased expressionin HO-1 cells.

No or only a small change in expression is observed in HO-1 cellstreated with MEZ (10 ng/ml; 24 hours or 96 hours), IFN-β (2,000units/ml; 96 hours), phenyl butyrate (PB) (4 mM; treated for 24 hours, 4days or 7 days), mycophenolic acid (MPA) (3 μM; 96 hours), transretinoic acid (RA) (2.5 μM; 24 hours), MPA+MEZ (3 μM+10 ng/ml; 96hours), RA+MEZ (2.5 μM; 96 hours), actinomycin D (5 μg/ml for 2 hours,assayed after 24 hours), adriamycin (0.1 μg/ml; 24 hours), vincristine(0.1 μg/ml; 24 hours), TNF-α (100 units/ml; 24 hours) or VP-16 (5 μg/ml;24 hours).

mda-4 is a novel gene which displays the following properties: 1) it isinducible in HO-1 cells during terminal differentiation (treatment withIFN-β+MEZ for 96 hours) and following 96 hour treatment with recombinantgamma interferon (alone or in combination with IFN-β); 2) it isinducible by IFN-β+MEZ in a series of human melanomas in addition toHO-1, normal cerebellum and GBM cells, but it is not expressed orinducible by IFN-β+MEZ in normal skin fibroblasts or three differenttypes of carcinomas (colorectal, endometrial adenocarcinoma or prostatecarcinoma); and 3) increased expression is induced in HO-1 cells treatedwith specific DNA damaging agents (cis-platinum, gamma irradiation andUV irradiation).

This gene represents a cytokine-, DNA damage-, andchemotherapy-(cis-platinum) and terminal differentiation-inducible genepossibly restricted to cells of neuroectodermal origin (melanoma andcentral nervous system). mda-4 may prove useful: 1) as a marker forspecific tissue lineage's (i.e., neuroectodermal) (diagnosticapplications); 2) to monitor response to DNA damage (induced by gammairradiation and UV irradiation) and treatment with chemotherapeuticagents which work in a similar manner as cis-platinum (diagnosticapplications); 3) to assay for types I (IFN-α and IFN-β) and type II(IFN-γ) interferon in biological fluids (diagnostic applications); and4) to identify compounds which have the capacity to induce terminaldifferentiation in human melanoma cells (drug screening programs toidentify new chemotherapeutic agents). Once full-length cDNAs areisolated, this gene (used in a sense orientation in an appropriateexpression vector) may prove useful in inhibiting growth and inducingterminal differentiation in human melanomas and specific central nervoussystem tumors (GBM) (therapeutic applications). Antisense constructs ofspecific regions of this gene could also prove useful in preventingdamage to normal tissue (e.g., bone marrow) treated with differentiationinducing and specific chemotherapeutic and DNA damaging agents(therapeutic applications).

    mda-4                                                                         (SEQ ID No. 4)                                                                  TTCTTCTTTGTAAAAGTTTTTAATACACTGCTGAAAGATAAATTCATTCCA                            - AAGAGAATAATTATATAGCAAGATATTATCGGCACAGTGGTTTCTTAGAGG                         - TAAATAGCGCCTCACGTGTGTTAGATGCTGAATCTGACCAAA                           

Properties of mda-5

This cDNA is novel (it has sequence homology to a Homo sapiensputatively transcribed partial sequence; Accession number Z20545; fromthe UK-HGMP, MRC Human Genome Mapping Project Resource, Centre ClinicalResearch Centre, Watford Road, Harrow, Middlesex, HA1 England)

Expression in HO-1 Human Melanoma Cells

Increased expression after 24 hour treatment of HO-1 cells withrecombinant human fibroblast interferon (IFN-β) (2,000 units/ml), and toa greater extent with IFN-β+MEZ (2,000 units/ml+10 ng/ml) incombination.

Analysis of terminally differentiated HO-1 cells, i.e., HO-1 cellstreated with the combination of IFN-β+MEZ (2,000 units/ml+10 ng/ml) for96 hours, indicate continued increased expression in IFN-β+MEZ treatedHO-1 cells.

Enhanced expression of mda-5 is also observed, albeit to a lesserextend, in HO-1 cells treated for 96 hours with immune interferon(IFN-γ) (2,000 units/ml) and in 96 hours IFN-β+IFN-γ (1,000units/ml+1,000 units/ml) treated HO-1 cells (greater increased than withIFN-γ alone) (note: this combination of agents results in a similardegree of growth suppression in HO-1 cells as does IFN-β+MEZ. However,growth suppression is reversible with this combination of interferons,whereas it is irreversible with the combination of IFN-β+MEZ).

mda-5 represents a novel IFN-γ-inducible gene which also displaysincreased expression during terminal cell differentiation in HO-1 cells.This gene could prove useful as a marker for immune interferon responseand as a marker for terminal differentiation in human melanoma cells.

Expression in Additional Human Melanoma Cells

Increased expression of this gene occurs in HO-1, C8161, C8161/6.3 (aC8161 human melanoma cell clone containing an inserted normal humanchromosome 6: These cells are tumorigenic in nude mice, but unlikeparental C8161 cells they are non-metastatic), FO-1, LO-1, SH-1, WM278and WM239 human melanoma cells treated with IFN-β+MEZ for 24 hours. Thisgene is constitutively expressed in immortalized human melanocytesFM5169 (transformed by SV40). Some upregulation is observed in FM5169following IFN-β+MEZ treatment for 24 hours.

Expression of mda-5 is increased by IFN-β+MEZ in 7 additional humanmelanoma cells besides the human melanoma cell it was cloned from i.e.,HO-1. In addition, this gene is expressed in melanocytes and itsexpression is increased to a lesser degree than in most human melanomasfollowing a 24 hour treatment with IFN-β+MEZ.

Expression in Normal Cerebellum, a Central Nervous System Tumor(Glioblastoma Multiforme) (GBM) and Normal Skin Fibroblast Cell Lines

mda-5 is express de novo at low levels in normal cerebellum, but not inGBM or normal skin fibroblasts. However, expression is increased innormal cerebellum (>10-fold) and expression is induced in GBM (smallinduction) and normal skin fibroblasts (good induction) following a 24hour treatment with IFN-β+MEZ.

This gene is susceptible to modulation by IFN-β+MEZ in human cerebellum,GBM and normal human skin fibroblasts. Differential de novo andinducible expression is seen in normal cerebellum cells versus GBM, withnormal cerebellum displaying both higher de novo and inducibleexpression.

Expression in Colorectal Carcinoma (SW613), Endometrial Adenocarcinoma(HTB113) and Prostate Carcinoma (LNCaP)

mda-5 is not expressed de novo in colorectal carcinoma (SW613) andendometrial adenocarcinoma (HTB113), whereas it is expressed at lowlevels in prostate carcinoma (LNCaP).

Following a 24 hour treatment with IFN-β+MEZ, mda-5 expression isinduced at high levels in colorectal carcinoma (SW613) cells, but noexpression is seen in endometrial adenocarcinoma (HTB113).

In the case of human prostate (LNCaP), IFN-β+MEZ treatment for 24 hoursresults in a 2- to 3-fold increase in mRNA expression.

This gene displays a differential pattern of both de novo and inducibleexpression in different human carcinomas.

Effect of Various Treatment Protocols on Expression in HO-1 Cells

A 24 hour treatment with IFN-β (2,000 units/ml), IFN-α (2,000 units/ml),IFN-β+MEZ (2,000 units/ml+10 ng/ml) and IFN-α+MEZ (2,000 units/ml+10ng/ml) results in increased expression in HO-1 cells.

mda-5 is induced after 96 hour treatment with IFN-γ and IFN-β+IFN-γ.Highest level of expression is observed in HO-1 cells treated withIFN-β+MEZ for 24 or 96 hours. Very low induction after 96 hour treatmentwith IFN-β (2,000 units/ml).

No change in expression is observed in HO-1 cells treated with MEZ (10ng/ml) (24 or 96 hours), MPA (3 μM; 96 hours), RA (2.5 μM; 96 hours),MPA+MEZ (3 μM+10 ng/ml; 96 hours), RA+MEZ (2.5 μM+10 ng/ml; 96 hours),phenyl butyrate (PB) (4 mM PB for 24 hours, 4 days or 7 days),cis-platinum (0.1 μg/ml; 24 hours), gamma irradiation (treated with 3gray and analyzed after 24 hours), UV (10 joules/mm², assayed 24 hourslater), actinomycin D (5 μg/ml for 2 hours, assayed 24 hours later),adriamycin (0.1 μg/ml; 24 hours), vincristine (0.1 μg/ml; 24 hours),cis-platinum (0.1 μg/ml; 24 hours), TNF-α (100 units/ml; 24 hours) orVP-16 (5 μg/ml; 24 hours).

mda-5 is a novel gene which displays the following properties: 1) it isinducible during terminal differentiation (treatment with IFN-β+MEZ for96 hours) and following treatment for 96 hours with recombinant gammainterferon (alone or in combination with IFN-β); 2) treatment for 24hours with IFN-β+MEZ results in increased expression in all humanmelanomas tested and in an SV40-immortalized human melanocyte; 3) it ishighly inducible by IFN-β+MEZ within 24 hours in normal cerebellum andnormal skin fibroblast cells, but it is only weakly inducible in GBM; 4)it is differentially inducible in three different types of carcinomas(with induction greatest in colorectal, low induction in prostatecarcinoma and no induction in endometrial adenocarcinoma); and 5)increased expression is induced in HO-1 cells treated with both type Iinterferon (IFN-α and IFN-β) and type II interferon (IFN-γ) (IFN-β ismore effective than IFN-α when used at an equivalent dose in enhancingexpression of this gene).

This gene represents a cytokine- and terminal differentiation-induciblegene displaying increased expression in all melanomas, in selectcarcinomas, in normal skin fibroblasts and in both normal cerebellum andGBM. mda-5 may be useful: 1) as a marker for specific tissue lineagesand for distinguishing tumors of similar histotype (i.e., carcinomas)(diagnostic applications; 2) to monitor response to type I and IIinterferon treatment (diagnostic applications; and 3) to identifycompounds which have the capacity to induce terminal differentiation inhuman melanoma cells (drug screening programs to identify newdifferentiation-inducing agents). Once full-length cDNAs are isolated,this gene (used in a sense orientation in an appropriate expressionvector) may prove useful in inhibiting growth and inducing terminaldifferentiation in human melanomas and other classes of tumors(therapeutic applications).

    mda-5                                                                         (SEQ ID No. 5)                                                                  CTGCAAAAGAAGTGTGCCGACTATAAATAAATGGTGAAATCATCTGCAAAT                            - GTGGCCAGGCTTGGGGAACAATGATGGTGCACAAAGGCTTAGATTTGCCTT                         - GTCTCAAAATAAGGAATTTTGTAGTGGTTTCAAAATATCACAAGAACGTAC                         - AAGTGGTAGATACTATCACATTCACTGACTATCAGAGTCG                                    -  (Seq. ID No. 6)                                                                   ACAAACCAGTGATTCCCCTTCCTCAGATACTGGGACTAACAGCTTCACCTG                    - GTGTTGGAGGGGCCACGAAGCAAGCCAAAGCTGAAGAACACATTTTAAAAC                         - TATGTGCCTATCTTGATGCATTTACTATTAAAACTGTTAAAGAAAACCTTG                         - ATCAACTGAAAAACCAAATACAGGAGCATGCAAGAAGTTTGCCATTGCAGA                         - TGCAACCAGAGAAGATCCATTTAAAGAGAAACTTCTAGAAATAATGACAAG                         - GATTCAAACTTATTGTCAAATGAGTCCAATGTCAGATTTTGGACTC                       

Properties of mda-6

mda-6 is identical to WAF1, CIP1, SDI1 that encodes a Mr.21,000 protein(p21) that is an inhibitor of cyclin dependent kinases.

Expression on HO-1 Human Melanoma Cells

Increased expression after 24 hour treatment of HO-1 cells withrecombinant human fibroblast interferon (IFN-β) (2,000 units/ml), MEZ(10 ng/ml) and to the greatest extent with IFN-β+MEZ (2,000 units/ml+10ng/ml) in combination.

Analysis of terminally differentiated HO-1 cells, i.e., HO-1 cellstreated with the combination of IFN-β+MEZ (2,000 units/ml+10 ng/ml) for96 hours indicate continued increased expression in IFN-β+MEZ treatedHO-1 cells; and (b) no significant or reduced alteration in expressionafter 96 hour treatment in HO-1 cells treated with IFN-β (2,000units/ml), IFN-γ (2,000 units/ml), MEZ (10 ng/ml), mycophenolic acid(MPA) (3 μM) (induces growth suppression increased melanin synthesis andmorphology changes, but not terminal differentiation in HO-1 cells)trans retinoic acid (RA) (2.5 μM) (increases melanin synthesis andtyrosinase activity, but does not alter growth, morphology or induceterminal differentiation in human melanoma cells), IFN-β+IFN-γ (1,000units/ml+1,000 units/ml) (growth suppressive without inducing markers ofdifferentiation), MPA+MEZ (3 μM+10 ng/ml) (reversible growth suppressionand induction of differentiation markers in HO-1 cells) and RA+MEZ (2.5μM+10 ng/ml) (growth suppression and reversible induction ofdifferentiation markers without inducing terminal cell differentiation).

mda-6 represents a novel gene which is enhanced in HO-1 cells byIFN-β+MEZ (which induces terminal differentiation) but not to the sameextent by agents inducing growth suppression or various markers ofdifferentiation. It could prove useful as a marker for the induction ofterminal differentiation in human melanoma cells.

Expression in Additional Human Melanoma Cells

Variable increases in expression of this gene occur in HO-1, C8161,C8161/6.3 (a C8161 human melanoma cell clone containing an insertednormal human chromosome 6: These cells are tumorigenic in nude mice, butunlike parental C8161 cells they are non-metastatic), FO-1, LO-1, SH-1,WM278 and WM239 human melanoma cells treated with IFN-β+MEZ for 24hours. This gene is constitutively expressed in immortalized humanmelanocytes FM516-SV (transformed by SV40). Some upregulation is alsoobserved in FMS16-SV following IFN-β+MEZ treatment for 24 hours.

Expression of mda-6 is increased by IFN-β+MEZ in 7 additional humanmelanoma cells besides the human melanoma cell it was cloned from, i.e.,HO-1. In addition, this gene is expressed in melanocytes and itsexpression is increased following a 24 hour treatment with IFN-β+MEZ.

Expression in Normal Cerebellum, a Central Nervous System Tumor(Glioblastoma Multiforme) (GBM) and Normal Skin Fibroblast Cell Lines

mda-6 is expressed de novo at high levels in normal cerebellum andnormal skin fibroblasts. mda-6 is not expressed at significant levels inGBM cells. mda-6 expression is increased in normal cerebellum (>10-fold)and expression is induced in GBM following a 24 hour treatment withIFN-β+MEZ. mda-6 expression is not altered in normal human skinfibroblasts after a 24 hour treatment with IFN-β+MEZ.

This gene is susceptible to modulation by IFN-β+MEZ in human cerebellumand GBM. In contrast, this gene is expressed and no change in expressionis seen following treatment in normal human skin fibroblasts.Differential de novo and inducible expression is also apparent in normalcerebellum cells versus GBM, with normal cerebellum displaying higher denovo expression. This gene could be a component of growth control incentral nervous system glial cells (including normal cerebellum cells),which is repressed in malignant GBM cells.

Expression in Colorectal Carcinoma (SW613), Endometrial Adenocarcinoma(HTB112) and Prostate Carcinoma (LNCaP)

mda-6 is expressed at high levels de novo in colorectal carcinoma(SW613) and prostate carcinoma (LNCaP). mda-6 de novo expression inendometrial adenocarcinoma (HTB113) is low. Treatment for 24 hours withIFN-β+MEZ does not significantly alter mda-6 expression in colorectalcarcinoma (SW613) or prostate carcinoma (LNCaP). Treatment for 24 hourswith IFN-β+MEZ induces mda-6 expression in endometrial adenocarcinoma(HTB113) to a similar level as in colorectal and prostate carcinomas.

mda-6 displays a differential pattern of both de novo and inducibleexpression in different human carcinomas. De novo expression is low inendometrial adenocarcinoma and high in colorectal and prostatecarcinoma. Inducible expression following treatment with IFN-β+MEZ isonly observed in endometrial adenocarcinoma cells.

Effect of Various Treatment Protocols on Expression in HO-1 Cells

Treatment with IFN-β (2,000 units/ml; 24 hours), MEZ (10 ng/ml; 24hours), IFN-β+MEZ (2,000 units/ml+10 ng/ml; 24 hours), actinomycin D (5μg/ml; 2 hour treatment followed by 24 hour growth), adriamycin (0.1μg/ml; 24 hour) and VP-16 (5 μg/ml; 24 hour) results in increasedexpression in HO-1 cells. Highest level of induction observed in HO-1cells treated with IFN-β+MEZ for 24 hours or 96 hours; and actinomycinD, adriamycin and VP-16 treated for 24 hours.

Decreased expression of mda-6 is observed in HO-1 cells treated withphenyl butyrate (PB) (4 mM) (24 hours, 4 or 7 days), cis-platinum (0.1μg/ml; 24 hours), UV (10 joules/mm² ; 2 hours after treatment), gammairradiation (3 gray; assayed after 24 hours), IFN-α (2,000 units/ml; 24hours) and TNF-α (100 units/ml; 24 hours).

No change in mda-6 expression observed in HO-1 cells treated with UV (10joules/mm²) and assayed after 14 or 24 hours and in HO-1 cells treatedfor 24 hours with IFN-α+MEZ (2,000 units/ml+10 ng/ml), or vincristine(0.1 μg/ml; 24 hours).

mda-6 is a novel gene which displays the following properties: 1) itsexpression is increased during terminal differentiation (treatment withIFN-β+MEZ for 96 hours); 2) treatment for 24 hours with IFN-β+MEZresults in variable increases in its expression in all human melanomastested and in an SV40-immortalized human melanocyte; 3) it is expressedin early stage melanomas (radial and early vertical growth phasemelanomas), but not or at reduced levels in more advanced melanomas(metastatic melanomas); 4) it is expressed de novo and highly inducibleby IFN-β+MEZ within 24 hours in normal cerebellum; 5) it is notexpressed de novo in GBM and only marginally induced in GBM after 24hour treatment with IFN-β+MEZ; 6) high levels of de novo expression areseen in normal skin fibroblasts, colorectal carcinoma (SW613) andprostate carcinoma (LNCaP), but IFN-β+MEZ treatment does notsignificantly alter expression; 7) endometrial adenocarcinoma (HTB113)cells display low levels of expression of this gene, whereas IFN-β+MEZtreatment for 24 hours results in high levels of expression; 8)expression is increased in HO-1 cells treated with actinomycin D,adriamycin and VP-16; 9) expression is reduced in HO-1 cells treatedwith phenyl butyrate, gamma irradiation, cis-platinum and TNF-α; and 10)expression is highest in normal melanocytes and a dysplastic nevus andreduced in radial growth phase (RGP) and vertical growth phase (VGP)primary melanomas and lowest in metastatic melanoma; 11) expression isreduced as a function of tumorigenic progression in Matrigel-progressedRGP and early VGP primary melanoma: 12) expression is low in tumorigenicand metastatic C8161 human melanoma cells and increased in threeindependent C8161 clones containing an inserted normal chromosome 6 thatare tumorigenic but not metastatic: 13) an immediate early response genei.e., induced in the presence of cyclohexmide, in human promyelocyticleukemia. HL-60 cells induced to differentiate into monocytes andmacrophages (treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA)or vitamin D3) or granulocytes (treatment with all-trans retinoic acid(RA) or dimethyl sulfoxide (DMSO)); 14) induced as a function of growtharrest and differentiation in human neuroblastoma cells by treatmentwith the combination of phenylacetate and RA; and 15) induced during theinduction of differentiation and growth arrest in human histiocyticlymphoma, U-937, cells by treatment with TPA.

mda-6 represents a terminal differentiation-regulated gene displayingincreased expression in all melanomas tested, in specific carcinomas, innormal cerebellum cells and in GBM cells treated with IFN-β+MEZ. mda-6is also induced during the induction of monocyte/macrophage andgranulocyte differentiation in human promyelocytic leukemia (HL-60)cells; differentiation in human neuroblastoma cells; and differentiationin histiocytic lymphoma (U-937) cells. This gene also displays increasedexpression in cells treated with specific chemotherapeutic agents,including adriamycin and VP-16. In contrast, expression of mda-6 isdecreased following treatment-with gamma irradiation, the demethylatinganticancer agent phenyl butyrate, the cytokine TNF-α and thechemotherapeutic agent cis-platinum. mda-6 may be useful: 1) as a markerfor specific tissue lineages and for distinguishing tumors of similarhistotype (i.e., carcinomas, astrocytomas) (diagnostic applications); 2)to monitor response to topoisomerase inhibitors such as VP-16 andspecific chemotherapeutic agents which function in a similar manner asadriamycin and cis-platinum (drug screening programs to identify newchemotherapeutic agents); 3) to identify compounds which have thecapacity to induce terminal differentiation in human melanoma cellsmyeloid leukemic cells, histiocytic lymphoma and neuroblastoma (drugscreening programs to identify new differentiation-inducing andchemotherapeutic agents); and 4) to monitor states of tumor progression,i.e., only expressed or expressed at higher levels in less aggressiveand early stage cancers. Once full-length cDNAs are isolated, this gene(used in a sense orientation in an appropriate expression vector) mayalso prove useful in inhibiting growth and inducing terminaldifferentiation in human melanomas and other classes of tumors(therapeutic applications). Overexpression of this gene in specific celltypes (such as bone marrow cells) may also result in a decreasedsensitivity of these cells to various DNA damaging agents andchemotherapeutic agents (therapeutic applications). This could proveuseful in protecting bone marrow cells from damage induced by radiationand chemotherapy (therapeutic applications). Similarly, use of antisenseconstructs may also result in a decreased sensitivity to growthsuppression in normal cells induced by specific classes of DNA damagingand therapeutic agents (therapeutic applications). This gene may alsoprove useful in the classification of more advanced astrocytomas (suchas GBM) from less advanced earlier stages of astrocytomas (diagnosticapplications). This gene may also prove useful in distinguishing betweenearly stage (early radial growth phase, early vertical growth phase)melanoma and late stage (late vertical growth phase, metastatic)melanoma (diagnostic applications).

    mda-6                                                                         (SEQ ID No. 7)                                                                  ATGCCACGTGGGCTCATATGGGGCTGGGAGTAGTTGTCTTTCCTGGCACTA                            - ACGTTGAGCCCCTGGAGGCACTGAAGTGCTTAGTGTACTTGGAGTATTGGG                         - GTCTGACCCAAACACCTTCCAGCTCCTGTAACATACTGGCCTGGACTGTTT                         - TCTCTCGCGCCTCCCCATGTGCTCCTGGTTCCCGTTTCCTCCACCTAGACT                         - GTAAACCTCTCGCA                                                              -  (Seq. ID No. 8)                                                                   CCTGCAGTCCTGGAAGCGCGAGGGCCTCAAACGCGCTCTACATCTTCTGCC                    - TTAGTCTCAGTTTGCGTGTCTTAATTATTATTTGTGTTTTAATTTAAACAC                         - CTCCTCATGTACATACCCTGGCCGCCCCCTGCCCCCCAGCCTCTCGGATTA                         - GAATTATTTAAACAAAAACTAGGCGGTTGAATGAGAGGTTCCTATGAGTAC                         - TGGGCATTTTTATTTTATGAAATACTATTTAAAGCCTCCTCATCCCATGTT                         - CTCCTTTTCCTCTCTCCCGGAGTT                                             

Properties of mda-7

mda-7 is a novel cDNA (it has no sequence homology with previouslyreported genes in the various DNA data bases).

Expression in HO-1 Human Melanoma Cells

Increased expression of mda-7 after 24 hour treatment of HO-1 cells withrecombinant human fibroblast interferon (IFN-β) (2000 units/ml), MEZ (10ng/ml) and to the greatest extent with IFN-β+MEZ (2000 units/ml+10ng/ml).

Increased expression of mda-7 is observed in HO-1 cells treated for 96hours with IFN-β (2000 units/ml), MEZ (10 ng/ml), MPA (3 μM),IFN-β+IFN-γ (1000 units/ml+1000 units/ml), IFN-β+MEZ (2000 units/ml+10ng/ml), MPA+MEZ (3 μM+10 ng/ml) and RA+MEZ (2.5 μM+10 ng/ml). Maximuminduction is observed with IFN-β+MEZ followed by MPA+MEZ andIFN-β+IFN-γ.

The relative level of mda-7 induction correlates with the degree ofgrowth suppression observed HO-1 cells treated with the various growthand differentiation modulating agents. The greatest increase inexpression is observed in cells induced to irreversibly loseproliferative capacity and become terminally differentiated by treatmentwith IFN-β+MEZ.

Expression in Additional Human Melanoma Cells

Increased expression of mda-7 occurs in HO-1, C8161, C8161/6.3 (a C8161human melanoma cell clone containing an inserted normal human chromosome6: These cells are tumorigenic in nude mice, but unlike parental C8161cells they are non-metastatic), FO-1, LO-1, SH-1, WM278 and WM239 humanmelanoma cells treated with IFN-β+MEZ for 24 hours. This gene isconstitutively expressed in immortalized human melanocytes FM5169(transformed by SV40). However, no increase in expression is observed inFM5169 following IFN-β+MEZ treatment for 24 hours.

mda-7 is either variably expressed or variably induced in all humanmelanoma cells treated with IFN-β+MEZ. In contrast, although this geneis expressed in melanocytes, no change in expression is observedfollowing a 24 hour treatment with IFN-β+MEZ.

Expression in Normal Cerebellum, a Central Nervous System Tumor(Glioblastoma Multiforme) (GBM) and Normal Skin Fibroblast Cell Lines

mda-7 is not expressed de novo in normal cerebellum, GBM or normal skinfibroblasts.

Expression of mda-7 is induced in normal cerebellum, GBM and normal skinfibroblasts following a 24 hour treatment with IFN-β+MEZ.

mda-7 is not expressed de novo but is susceptible to induction byIFN-β+MEZ in human cerebellum, GBM and normal human skin fibroblasts.

Expression in Colorectal Carcinoma (SW613), Endometrial Adenocarcinoma(HTB113) and Prostate Carcinoma (LNCaP)

mda-7 is not expressed de novo in colorectal carcinoma (SW613),endometrial adenocarcinoma (HTB113) or prostate carcinoma (LNCaP).

mda-7 is not induced in colorectal carcinoma (SW613), endometrialadenocarcinoma (HTB113) or prostate carcinoma (LNCaP) cells following a24 hour treatment with IFN-β+MEZ.

This gene is neither expressed de novo or inducible by IFN-β+MEZ inhuman carcinomas.

Effect of Various Treatment Protocols on Expression in HO-1 Cells

Treatment with IFN-β (2000 units/ml; 24 hours), MEZ (10 ng/ml; 24hours), IFN-β+MEZ (2000 units/ml+10 ng/ml; 24 hours and 96 hours),IFN-α+MEZ (2000 units/ml+10 ng/ml; 24 hours), adriamycin (0.1 μg/ml; 24hours), vincristine (0.1 μg/ml; 24 hours), and UV (10 joules/mm² andassayed 24 hours later) results in increased mda-7 expression in HO-1cells. mda-7 is also induced after 96 hour treatment with MPA (3 μM),IFN-β+IFN-γ (1000 units/ml+1000 units/ml), MPA+MEZ (3 μM+10 ng/ml) andRA+MEZ (2.5 μM+10 ng/ml). Highest level of expression observed in HO-1cells treated with IFN-β+MEZ for 24 or 96 hours.

No induction in mda-7 expression is observed in HO-1 cells treated withIFN-α (2000 units/ml; 24 hours), IFN-γ (2000 unit/ml; 96 hours), phenylbutyrate (4 mM PB for 24 hours, 4 days or 7 days), cis-platinum (0.1μg/ml; 24 hours), gamma irradiation (treated with 3 gray and analyzedafter 24 hours), actinomycin D (5 μg/ml for 2 hours, assayed 24 hourslater), TNF-α (100 units/ml; 24 hours) or VP-16 (5 μg/ml; 24 hours).

mda-7 is a growth and differentiation and sensescence-regulated novelgene which displays the following properties: 1) it is inducible duringterminal differentiation (treatment with IFN-β+MEZ for 96 hours) andfollowing treatment for 96 hours with many growth modulating anddifferentiation inducing agents; 2) treatment for 24 hours withIFN-β+MEZ results in increased expression in all human melanomas tested,but not in an SV40-immortalized human melanocyte; 3) it is not expressedde novo but it is highly inducible by IFN-β+MEZ within 24 hours innormal cerebellum, GBM and normal skin fibroblast cells; 4) it is notexpressed or inducible in colorectal, endometrial or prostatecarcinomas; 5) increased expression is induced in HO-1 cells treatedwith adriamycin, vincristine and UV irradiation; and 6) it is notexpressed in growing human neuroblastoma cells but it is induciblefollowing growth suppression and the induction of terminaldifferentiation; 7) it is not expressed in human promyeloctyic leukemia(HL-60 and human histiocytic lymphoma (U-937) cells but it is inducedfollowing the induction of growth arrest and terminal differentiation;and 8) it is not expressed in actively growing human cells but it isinduced during cellular senescence.

mda-7 is a novel growth- and terminal differentiation-regulatable genedisplaying increased expression in all melanomas (but not inmelanocytes), and in normal skin fibroblasts and in both normalcerebellum and GBM cells treated with IFN-β+MEZ. In contrast, mda-7 isnot expressed or induced in a series of carcinomas. mda-7 may beuseful: 1) as a marker for specific tissue lineage's (i.e., melanomasfrom keratinocytes) (diagnostic applications); 2) in distinguishingfibroblasts (inducible with IFN-β+MEZ) from carcinomas (non-induciblewith IFN-β+MEZ) (diagnostic applications); 3) for the identification ofagents capable of inducing growth suppression and various components ofthe differentiation process (including terminal differentiation) inhuman melanomas (drug screening programs to identify newdifferentiation-inducing and chemotherapeutic agents); and 4)distinguishing melanocytes, and perhaps nevi, from early and late stagemelanoma cells (diagnostic applications). Once full-length cDNAs areisolated, this gene (used in a sense orientation in an appropriateexpression vector) may also prove useful in inhibiting growth andinducing terminal differentiation in human melanomas (therapeuticapplications).

    mda-7                                                                           (SEQ ID No. 9)                                                                    CAGAATATTGTGCCCCATGCTTCTTTACCCCTCACAATCCTTGCCACAGTG                        - TGGGCAGTGGATGGGTGCTTAGTAAGTACTTAATAAACTGTGGTGCTTTTT                         - TTGGCCTGTCTTTGGATTGTTAAAAAACAGAGAGGGATGCTTGGATGTAAA                         - CTGAACTTCAGAGCTGAAATCACACTGTCTCTGATATCT                              

Properties of mda-8

mda-8 is a novel cDNA (it has no sequence homology with previouslyreported genes in the various DNA data bases)

Expression in HO-1 Human Melanoma Cells

Increased expression of mda-8 results in HO-1 cells after 24 hourtreatment with the combination of IFN-β+MEZ (2000 units/ml+10 ng/ml).

Analysis of terminally differentiated HO-1 cells, i.e., HO-1 cellstreated with the combination of IFN-β+MEZ (2000 units/ml+10 ng/ml) for96 hours indicate continued increased expression of mda-8.

Treatment of HO-1 cells for 96 hours with immune interferon (IFN-γ)(2000 units/ml) or IFN-β+IFN-γ (1000 units/ml+1000 units/ml) results inenhanced mda-8 expression. The level of increased mda-8 expression at 96hour is similar in IFN-γ, IFN-β+IFN-γ and IFN-β+MEZ treated HO-1 cells.(Note: The combination of IFN-β+IFN-γ results in a similar degree ofgrowth suppression at 96 hour in HO-1 cells as does IFN-β+MEZ. However,growth expression is reversible with the combination of interferons,whereas it is irreversible with the combination of IFN-β+MEZ).

mda-8 is a novel IFN-γ-inducible gene which also displays increasedexpression during terminal cell differentiation in HO-1 human melanomacells. mda-8 could prove useful as a marker for immune interferonresponse and a marker for terminal differentiation in human melanomacells.

Expression in Additional Human Melanoma Cells

Increased expression of mda-8 occurs in HO-1, C8161 and WM278 humanmelanoma cells treated for 24 hours with IFN-β+MEZ (2000 units/ml+10ng/ml).

No change in expression of mda-8 is seen in additional human melanomastreated for 24 hours with IFN-β+MEZ, including C8161/6.3 (a C8161 humanmelanoma cell clone containing an inserted normal human chromosome 6:These cells are tumorigenic in nude mice, but unlike parental C8161cells they are non-metastatic), FO-1, LO-1, SH-1, and WM239.

Expression of this gene is increased by IFN-β+MEZ in specific humanmelanoma cells.

Expression in Normal Cerebellum, a Central Nervous System Tumor(Glioblastoma Multiforme) (GBM) and Normal Skin Fibroblast Cell Lines

mda-8 is expressed de novo in normal cerebellum, but not in GBM.

mda-8 is expressed de novo in normal skin fibroblasts.

Growth for 24 hours in IFN-β+MEZ (2000 units/ml+10 ng/ml) results inmarginal changes in mda-8 expression in normal cerebellum and normalskin fibroblasts.

Expression of mda-8 is induced at high levels in GBM cells following a24 hour exposure to IFN-β+MEZ.

This gene is expressed de novo in both normal cerebellum and normal skinfibroblasts, but not in GBM. This gene is induced by IFN-β+MEZ in humanGBM, but expression is not altered in normal cerebellum cells and normalskin fibroblasts.

Expression in Colorectal Carcinoma (SW613), Endometrial Adenocarcinoma(HTB113) and Prostate Carcinoma (LNCaP)

mda-8 is expressed de novo in colorectal carcinoma (SW613), endometrialadenocarcinoma (HTB113) and prostate carcinoma (LNCaP).

Following a 24 hour treatment with IFN-β+MEZ, expression of mda-8 isunaffected in colorectal carcinoma (SW613), endometrial adenocarcinoma(HTB113) and prostate carcinoma (LNCaP) cells.

mda-8 is expressed de novo in the three types of carcinomas. mda-8 geneexpression is not altered in the three carcinomas after treatment for 24hours with IFN-β+MEZ.

Effect of Various Treatment Protocols on Expression in HO-1 Cells

Increased expression of mda-8 results after treatment with IFN-β (2000units/ml; 24 hours), actinomycin D (5 μg/ml for 2 hours, assayed 24hours later), adriamycin (0.1 μg/ml; 24 hours), cis-platinum (0.1 μg/ml;24 hours) and UV (10 joules/mm², assayed 2, 14 and 24 hours later).

A 96 hour treatment with IFN-γ (2000 units/ml), IFN-β+MEZ (2000units/ml) and IFN-β+IFN-γ (1000 units+1000 units) results in increasedmda-8 expression.

No change in expression of mda-8 is observed in HO-1 cells treated withMEZ (10 ng/ml; 24 or 96 hours), IFN-β (2000 units/ml; 24 or 96 hours),MPA (3 μM; 96 hours), RA (2.5 μM; 96 hours), MPA+MEZ (3 μM+10 ng/ml; 96hours), RA+MEZ (2.5 μM+10 ng/ml), phenyl butyrate (4 mM PB for 24 hours,4 days or 7 days), gamma irradiation (treated with 3 gray and analyzedafter 24 hours), vincristine (0.1 μg/ml; 24 hours), TNF-α (100 units/ml;24 hours), VP-16 (5 μg/ml; 24 hours), IFN-α (2000 units/ml) or IFN-α+MEZ(2000 units/ml+10 ng/ml).

mda-8 is a novel gene which displays the following properties: 1) it isinducible during terminal differentiation (treatment with IFN-β+MEZ for96 hours) and following treatment for 96 hours with recombinant gammainterferon (alone or in combination with IFN-β); 2) treatment for 24hours with IFN-β+MEZ results in increased expression in only selecthuman melanomas; 3) it is expressed de novo in normal cerebellum, normalskin fibroblasts, colorectal carcinoma (SW613), endometrialadenocarcinoma (HTB113) and prostate carcinoma (LNCaP), but not in GBM;4) treatment with IFN-β+MEZ for 24 hours results in no change inexpression in normal cerebellum, normal skin fibroblasts, colorectalcarcinoma (SW613), endometrial adenocarcinoma (HTB113) and prostatecarcinoma (LNCaP); 5) treatment for 24 hours with IFN-β+MEZ inducesexpression in GBM cells; and 6) increased expression is induced in HO-1cells treated with actinomycin D, adriamycin, cis-platinum and UVirradiation.

mda-8 is a cytokine- and terminal differentiation-responsive genedisplaying increased expression in specific human melanomas and GBMcells treated with IFN-β+MEZ (also inducible in HO-1 after 96 hourtreatment with IFN-γ and IFN-γ+IFN-β). Enhanced expression is alsoapparent in HO-1 human melanoma cells treated with the transcriptioninhibitor actinomycin D, the chemotherapeutic agents adriamycin andcis-platinum and UV irradiation. mda-8 may be useful: 1) as a marker fordistinguishing between normal glial cells and malignant astrocytomas(such as GBM) (diagnostic applications); 2) to monitor response to typeII interferon treatment (diagnostic applications); and 3) to identifycompounds which have the capacity to induce terminal differentiation,induce similar cytotoxic effects as adriamycin, cis-platinum and UVirradiation (drug screening programs to identify newdifferentiation-inducing and chemotherapeutic agents). Once full-lengthcDNAs are isolated, this gene (used in a sense orientation in anappropriate expression vector) may prove useful in inhibiting growth andinducing terminal differentiation in specific human melanomas andglioblastoma multiforme tumors (therapeutic applications).

    mda-8                                                                           (SEQ ID No. 10)                                                                   TTAAAGTTTGCCCTTGTGCTAAAGTGCCAGTGTATGTATGTTATACTTGAT                        - TTGGTTGTAAACTATATTTCAAAGTAAACCCTAGTGTAATAAGTTTTATAA                         - CTAAAAAGGTTTAAGCTGCTAAAACTATTTTTAAGAGATGTGAAATCGAGT                         - ATGGGACTATCTTTTTTTCCTCCTCTAAA                                        

Properties of mda-9

mda-9 is a novel cDNA (it displays sequence homology to humantransforming growth factor-β (TGF-β) mRNA, 55.1% homology in 138 bp;GB-Pr:Humtgfbc).

Expression in HO-1 Human Melanoma Cells

Increased expression of mda-9 occurs after 24 hour treatment of HO-1cells with the combination of IFN-β+MEZ (2000 units/ml+10 ng/ml).

Increased expression of mda-9 persists in terminally differentiated HO-1cells, i.e., HO-1 cells treated with the combination of IFN-β+MEZ (2000units/ml+10 ng/ml) for 96 hours.

mda-9 is a novel gene (with homology to TGF-β) which displays increasedexpression in terminally differentiated HO-1 human melanoma cells.

Expression in Additional Human Melanoma Cells

Variable increases in expression of mda-9 occurs in HO-1 and C8161 humanmelanoma cells treated for 24 hours with IFN-β+MEZ (2000 units/ml+10ng/ml).

The level of expression of mda-9 decreases in SH-1 cells treated for 24hours with IFN-β+MEZ (2000 units/ml+10 μg/ml).

No change in mda-9 expression results in FO-1, LO-1 or C8161/6.3 cells(a C8161 human melanoma cell clone containing an inserted normal humanchromosome 6: These cells are tumorigenic in nude mice, but unlikeparental C8161 cells they are non-metastatic).

Expression of mda-9 is increased by IFN-β+MEZ in specific human melanomacells. The lack of enhanced expression in C8161/6.3 cells treated withIFN-β+MEZ, whereas parental C8161 cells do show an increase, suggeststhat modulation of this gene may correlate with more advanced stages ofmelanoma development (i.e., melanoma cells with metastatic potential).

Effect of Various Treatment Protocols on Expression in HO-1 Cells

Increased expression of mda-9 is observed in HO-1 cells treated withIFN-β (2000 units/ml; 24 hours), MEZ (10 ng/ml; 24 hours), IFN-β+MEZ(2000 units/ml+10 ng/ml; 24 and 96 hours), phenyl butyrate (4 mM PB for24 hours, 4 days or 7 days), gamma irradiation (treated with 3 gray andanalyzed after 24 hours), TNF-α (100 units/ml; 24 hours), IFN-α (2000units/ml), IFN-α+MEZ (200 units/ml+10 ng/ml), VP-16 (5 μg/ml; 24 hours)or UV (10 joules/mm², assayed 2 or 14 hours later).

No change in mda-9 expression is observed in HO-1 cells treated withactinomycin D (5 μg/ml for 2 hours, assayed 24 hours later), UV (10joules/nm², assayed 24 hours later), cis-platinum (0.1 μg/ml; 24 hours),vincristine (0.1 μg/ml; 24 hours), IFN-β (2000 units/ml; 96 hours),IFN-γ (2000 units/ml; 96 hours), MEZ (10 ng/ml; 96 hours), MPA (3 μM; 96hours), RA (2.5 μM; 96 hours), IFN-β+IFN-γ (1000 units/ml+1000 units/ml;96 hours), MPA+MEZ (3 μM+10 ng/ml; 96 hours) or RA+MEZ (2.5 μM+10ng/ml).

mda-9 is a novel gene with sequence homology to TGF-β which displays thefollowing properties: 1) it is inducible during terminal differentiation(treatment with IFN-β+MEZ for 96 hours) in HO-1 human melanoma cells; 2)treatment for 24 hours with IFN-β+MEZ results in increased expression inseveral human melanomas; 3) treatment for 24 hours with IFN-β+MEZresults in increased expression in the tumorigenic and metastatic humanmelanoma C8161, but not in C8161/6.3 which is tumorigenic but notmetastatic; and 4) increased expression is induced in HO-1 cells treatedwith a number of agents including phenyl butyrate, gamma irradiation,TNF-α, UV irradiation (after 2 and 14 hours, but not after 24 hours),IFN-α and IFN-α+MEZ.

mda-9 is a terminal differentiation-responsive gene displaying increasedexpression in several human melanomas treated with IFN-β+MEZ. Enhancedexpression is induced by IFN-β+MEZ in the tumorigenic and metastatichuman melanoma cell C8161, but not its reverted derivative C8161/6.3(which retains tumorigenicity, but has lost metastatic potential).Increased expression is also apparent in HO-1 human melanoma cellstreated with the demethylating anticancer agent phenyl butyrate, thecytokine TNF-α, gamma irradiation and UV irradiation.

mda-9 may be useful: 1) as a marker for distinguishing between earlystage and more progressed human melanoma (diagnostic applications); and2) to identify compounds which have the capacity to induce terminaldifferentiation and to induce specific patterns of DNA damage as inducedby UV irradiation and gamma irradiation (drug screening programs toidentify new differentiation-inducing and chemotherapeutic agents). Oncefull-length cDNAs are isolated, this gene (used in a sense orientationin an appropriate expression vector) may also prove useful in inhibitinggrowth and inducing terminal differentiation in specific human melanomas(therapeutic applications). When used in an antisense orientation,expression of this gene might allow normal cells (such as bone marrowcells) to be engineered to be resistant to cytotoxicity induced byspecific chemotherapeutic agents and gamma irradiation (therapeuticapplications).

    mda-9                                                                           (SEQ ID No. 11)                                                                   AAAACTTTCAAGAGATTTACTGACTTTCCTAGAATAGTTTCTCTACTGGAA                        - ACCTGATGCTTTTATAAGCCATTGTGATTAGGATGACTGTTACAGGCTTAG                         - CTTTGTGTGAAAACCAGTCACCTTTCTCCTAGGTAATGAGTAGTGCTGTTC                         - ATATTACTTTAGTTCTATAGCATACTCGATCTTTAACATGCTATCATAGTA                         - CATTAGATGATG                                                         

Additional mda Genes Isolated Using Subtraction Hybridization from HO-1Human Melanoma Cells Treated with IFN-β+MEZ

mda-1: Novel gene which displays increased expression in IFN-β andIFN-β+MEZ treated HO-1 cells after 24 hours (HP 2-36). (Jiang andFisher, Molecular and Cellular Differentiation, 1 (3), in press, 1993).

mda-2: Novel gene which displays increased expression in IFN-β andIFN-β+MEZ treated HO-1 cells after 24 hours (HP-3-31). (Jiang andFisher, Molecular and Cellular Differentiation, 1 (3), in press, 1993).

mda-3: Increased expression in MEZ and IFN-β+MEZ treated HO-1 cellsafter 24 hours (HP 2-4). (Identical to Human GOS 19-1 mRNA, cytokine(Gb-Pr:Hummipla), human TPA-inducible mRNA, pLD78 (GB-Pr:Humpld78).(Jiang and Fisher, Molecular and Cellular Differentiation, 1 (3), inpress, 1993).

mda-11: Novel gene which displays increased expression in IFN-β+MEZtreated HO-1 cells after 24 hours. (HJ 2-78). (87.2% identity to the ratribosomal protein IF116).

    (Seq. ID No. 2)                                                                 CGCACGTCACCCACCTTCCGGCGGCCGAAGACACTGCGACTCCGGAGACAG                            - CCCAAATATCCTCGGAAGAGCGCTCCCAGGAGAAACAAGCTTGACCACTAT                         - GCTATCATCAAGTTTCCGCTGACCACTGAGTCTGCCATGAAGAAGATAGAA                         - GACAACAACACACTTGTGTTCATTGTGGATGTTAAAGCCAACAAGCACCAG                         - ATTAACAGCTGTGAGAGCTGTATGACATTGATGTGCAGTACACCTGATCGT                        CT                                                                      

mda-12: Gene which displays increased expression in IFN-β+MEZ treatedHO-1 cells after 24 hours. (HP 3-8). (Identical to Human GOS19-3 mRNA(Gb-Humcpgcus2), LD78A (Gb-Pr:Humld78a).

mda-13: Gene which displays increased expression in IFN-β and IFN-β+MEZtreated HO-1 cells after 24 hours. (HP S-7). (Identical to interferonstimulated gene -56 (ISG56), an IFN-β inducible gene).

mda-14: Gene which displays increased expression in IFN-β+MEZ treatedHO-1 cells after 24 hours. (HP 2-59 and HP 3-114, same gene isolatedindependently two times). (Identical to interleukin-8 (IL-8)(Gb-Un:M28130), human mRNA for MDNCF (monocyte derived neutrophilchemotactic factor) (Gb-Pr:Nummdncf).

    (Seq. ID No. 13)                                                                TAAAAAAATTCATTCTCTGTGGTATCCAAGAATCAGTGAAGATGCCAGTGA                            - AACTTCAAGCAAATCTACTTCAACACTTCATGTATTGTGTGGGTCTGTTGT                         - AGGGTTGCCAGATGCAATACAAGATTCCTGGTTAAATTTGAATTTCAGTAA                         - ACAATGAATAGTTTTTCATTGTACATGAAATATCAGAACATACTTATATGT                         - AAGTATATTATTGATGACAAACACAATATTTAATATA                                

mda-15: Gene which displays increased expression in IFN-β+MEZ treatedHO-1 cells after 24 hours (HP 2-64). (Identical to vimentin,intermediate filament protein (Gb-Pr:Humviment).

mda-16: Gene which displays increased expression in IFN-β+MEZ treatedHO-1 cells after 24 hours. (HP 2-18). (Identical to human apoferritin Hgene (Gb-Pr:Humferg2).

mda-17: Gene which displays increased expression in IFN-β+MEZ treatedHO-1 cells after 24 hours. (HP 2-40). (Identical to IFP-53 (GbPr:Humifp), IFN-inducible gamma 2 protein (Gb-Huminfig).

    (Seq. ID No. 14)                                                                GGGGGTGAAACTTTCCAGTTTACTGAACTCCAGACCATGCATGTAGTCCAC                            - TCCAGAAATCATGCTCGCTTCCTTGGCACACAGTGTTCTCCTGCCAAATGA                         - CCCTAGACCCTCTGTCCTGCAGAGTCAGGGTGGCTTTTACCCTGACTGTGT                         - CGATGCAGAGTCTGCTCGACAGAT                                             

mda-18: Gene which displays increased expression to IFN-β+MEZ treatedHO-1 cells after 24 hours (HP 2-45). (Identical to hnRNP A1 protein(Gb-Pr:Humrnpa1), RNA binding protein (Gb-Pr:Humhnrnpa).

    (Seq. ID No. 15)                                                                TACGATCAGACTGTTACATTTAGCAATCAACAGCATGGGGCGAAAAAAAAA                            - AATCTACTTAAAACCCTTTGTTGGAATGCTTTACACTTTCCACAGAACAGA                         - AACTAAAATAACTGTTTACATTAGTCACAATACAGTCTCGA                            

Fifth Series of Experiments

The carcinogenic process often proceeds through a series of interrelatedstages and is regulated by multiple genetic changes and environmentalfactors (1-6). Although the specific events controlling each componentof this multistep process remain to be defined, a recurrent theme inmany cancer cells is an aberrant pattern of differentiation (7-10). Inaddition, as cancer cells evolve, ultimately developing new phenotypesor an increased expression of pre-existing transformation-relatedphenotypes, the degree of expression of differentiation associatedtraits is often further diminished. Malignant melanoma epitomizes theprocess of tumor progression and emphasizes the selective nature of themetastatic phenotype and the growth dominant properties of metastaticcells (11-14). Of the numerous types of cancer developing in NorthAmerican populations, melanoma is increasing at the fastest rate and itis estimated that as many as 1 in 100 currently born children mayeventually develop superficial spreading type melanoma (11). Althoughmelanoma is readily curable at early stages, surgical andchemotherapeutic interventions are virtually ineffective in preventingmetastatic disease and death in patients with advanced stages ofmalignant melanoma. These observations emphasize the need for improvedtherapeutic approaches to more efficaciously treat patients withmetastatic melanoma.

Development of malignant melanoma in humans, with the exception ofnodular type melanoma, consists of a series of sequential alterations inthe evolving tumor cells (11-15). These include conversion of a normalmelanocyte into a common acquired melanocyctic nevus (mole), followed bythe development of a dysplastic nevus, a radial growth phase (RGP)primary melanoma, a vertical growth phase (VGP) primary melanoma andultimately a metastatic melanoma (FIG. 20). Although readily treatableduring the early stages of development, even during the VGP if thelesion is ≦0.76-mm thick, currently employed techniques are not veryeffective (<20% survival) in preventing metastatic spread and morbidityin patients with VGP lesions >4.0-mm thickness (11). This exceptionalmodel system is ideally suited to evaluate the critical gene expressionchanges that mediate both the early and late phases of melanomaevolution.

A potentially less toxic approach to cancer therapy involves a processtermed differentiation therapy (7,9,10,16,17). Two premises underliethis therapeutic modality. (A) Many types of neoplastic cells displayaberrant patterns of differentiation resulting in unrestrained growth;and (B) Treatment with the appropriate agent(s) can result in thereprogramming of tumor cells to lose proliferative capacity and becometerminally differentiated. Intrinsic in this hypothesis is theassumption that the genes that mediate normal differentiation in manytumor cells are not genetically defective, but rather they fail to beexpressed appropriately. The successful application of differentiationtherapy in specific instances may result because the appropriate genesinducing the differentiated phenotype become transcriptionally activatedresulting in the production of necessary gene products required toinduce terminal cell differentiation. Applicants have tested thishypothesis using human melanoma cells (8,10,18-23). Treatment of humanmelanoma cells with the combination of recombinant human fibroblastinterferon (IFN-β) and the antileukemic compound mezerein (MEZ) resultsin a rapid cessation of growth, an induction of morphological changes,an alteration in antigenic phenotype, an increase in melanin synthesisand an irreversible loss in proliferative capacity, i.e., terminal celldifferentiation (18,21,23). In contrast, treatment of the same melanomacells with equivalent doses of either IFN-β or MEZ alone results inspecific differentiation-related and immunologically-related changes andgrowth suppression, but terminal differentiation does not occur(18,21,24-34).

This invention summarizes the results of applicants' analysis of theprocess of reversible and irreversible (terminal) differentiation inhuman melanoma cells. By using the technique of subtractionhybridization a series of novel genes, termed melanoma differentiationassociated (mda), have been identified that display enhanced expressionduring differentiation and growth arrest in human melanoma cells. Thesenewly identified mda genes should prove useful in defining the molecularbasis of human melanoma growth, differentiation and transformationprogression.

Dissecting the Processes of Growth Control and Differentiation in HumanMelanoma Cells

The process of terminal differentiation in HO-1 cells involves a numberof changes in cellular phenotype and gene expression (18,21,23,35).Biochemical and cellular changes include growth suppression, changes inmelanin synthesis (biochemical differentiation) and modified antigenicproperties (immunologic differentiation) (18,21,23,29,34,35). Theability to define the relationship between the different components ofgrowth and differentiation and the corresponding gene expression changesinduced in human melanoma cells has been assisted by the identificationof specific compounds that induce different components of theseprocesses (FIGS. 21A-H) (18,21,23,33,36).

Growth of HO-1 cells in the combination of IFN-β plus recombinant immuneinterferon (IFN-γ) results in a similar level of growth suppressionafter 96 hr as does the combination of IFN-β+MEZ (23,33). However, thiscombination of interferons induces reversible growth arrest and it doesnot cause an increase in melanin synthesis above that induced by IFN-βalone (33). This specific combination of agents permits a dissociationbetween reversible growth suppression and induction of irreversiblegrowth suppression and terminal differentiation. Treatment of HO-1 cellswith compounds such as all-trans retinoic acid (RA), mycophenolic acid(MPA), IFN-β and MEZ results in a reversible increase in melaninsynthesis, i.e., reversible biochemical differentiation (FIGS. 21A-H)(23,36). However, although MPA, IFN-β and MEZ induce growth suppression,RA increases melanin and tyrosinase levels without altering HO-1 growth(23,26]. These results indicate that RA can be used to identify geneexpression changes correlating directly with increased melanin synthesisin the absence of growth suppression. Additional compounds that haveproven of interest, include MPA and MEZ that induce morphologicdifferentiation in HO-1 cells (FIG. 22). These changes in HO-1 cells arealso reversible following growth in the absence of the inducing agent.The only currently available combination of agents that can irreversiblyinduce the spectrum of differentiation changes resulting in terminalcell differentiation in HO-1 cells is IFN-β+MEZ (18,21,23).

Gene Expression Changes Induced in Human Melanoma Cells DuringReversible Growth Suppression and Terminal Cell Differentiation

As discussed briefly above, treatment of HO-1 cells with a number ofagents, either independently or in combination, can result in reversiblegrowth suppression and the reversible expression ofdifferentiation-associated changes or terminal cell differentiation(23). A key question is the nature of the gene expression changes thatcorrelate with these modifications in HO-1 phenotype. To begin toaddress this issue, studies have focused on genes involved in earlystages of growth response (c-fos, c-iyc, c-jun, jun-B and jun-D), incytokine response (interferon stimulated gene-15 (ISG-15), ISG-54, HLAClass I antigen, HLA Class II antigen, melanoma growth stimulatoryactivity (gro/MGSA)), in the extracellular matrix (fibronectin andtenascin), as receptors for extracellular matrix proteins (α₅ integrinand β₁ integrin), and as cytoskeletal proteins β-actin and γ-actin)(23,25). The pattern of gene expression changes occurring in terminallydifferentiated HO-1 cells is shown in Table 3. Analysis of theexpression of similar genes in cells undergoing reversible growth arrestindicates that none of these changes are unique to terminallydifferentiated HO-1 cells (23,25). Instead, these specific changes ingene expression appear to correlate with various aspects of thedifferentiation and growth arrest program induced in HO-1 cells by thevarious compounds.

                  TABLE 3                                                         ______________________________________                                        Gene Expression Changes Observed in Terminally                                  Differentiated HO-1 Cells                                                                                   No                                              Increased Decreased Change in                                                 Expression Expression Expression                                            ______________________________________                                        c-jun          c-myc        c-fos                                               jun-B cyclin A RB                                                             HLA Class I cyclin B N-cadherin                                               ISG-15 tenascin                                                               ISG-54 γ-actin                                                          gro/MGSA β-actin                                                         α.sub.5 integrin cdc 2                                                  β.sub.1 integrin histone H1                                              fibronectin histone H4                                                      ______________________________________                                         HO-1 cells were grown for 96 hr in the presence of 2000 units/ml IFN.beta     and 10 ng/ml MEZ prior to isolation of total RNA and analysis of gene         expression by Northern blotting analysis (23). Cells remain viable under      these conditions, but they irreversibly lose proliferative ability            (18,23).                                                                 

Autocrine Factors Involved in Gene Expression Changes Induced in HumanMelanoma Cells During Reversible Growth Suppression and Terminal CellDifferentiation

A potentially important mediator of growth arrest during differentiationin hematopoietic cells is autocrine IFN-β (37-39). Evidence suggesting alink between autocrine IFN-β and differentiation in hematopoietic cellsinclude: (a) the observation that IFN-β neutralizing antibodies canpartially abrogate the reduction in c-myc levels and growth suppressionoccurring during hematopoietic differentiation; (b) the induction ofinterferon regulatory factor 1 (IRF-1) during myeloid differentiation;(c) the partial reversal by IRF-1 antisense oligomers of growthinhibition and the induction of differentiation induced in leukemiccells by interleukin-6 and leukemia inhibitory factor; and (d) theinduction of specific type I interferon (IFN-α/β) gene expression duringterminal differentiation in hematopoietic cells (37-39).

Enhanced expression of interferon responsive-genes and the gro/MGSA geneoccurs in HO-1 cells during the processes of reversible and irreversibledifferentiation (23). These observations suggest that autocrine-feedbackpathways could contribute to the gene expression changes observed duringthe differentiation process. To directly test for this possibility, HO-1cells were treated with various inducing agents for 24 hr, cultures werewashed free of inducers and then grown for 72 hr in medium lacking theinducing compounds. The conditioned medium from these cells wascollected and tested for its ability to induce gene expression changesin naive HO-1 cells (23). Conditioned medium obtained from cells treatedwith IFN-β+MEZ induced growth suppression and a number of geneexpression changes also apparent in inducer-treated HO-1 cells (23,40).These include, enhanced c-jun, α₅ integrin and fibronectin expressionand induction of jun-B, HLA Class I, ISG-15, and gro/MGSA expression(23). These observations suggest two autocrine loops may be associatedwith differentiation in HO-1 cells, one involving an autocrine IFN-β andthe other an autocrine gro/MGSA. Support for the IFN-β autocrine loop isindicated by the ability of IFN-β antibodies to partially neutralizeISG-15 induction by conditioned medium and the direct induction of theIFN-β gene as monitored by RT-PCR by conditioned medium (40). However,conditioned medium from IFN-β+MEZ-treated HO-1 cells does not induceterminal differentiation in HO-1 cells (40). Similarly, additionalagents that induce reversible growth arrest and differentiation alsoproduce conditioned medium that can induce type I interferon responsivegenes in naive HO-1 cells. These results suggest that specific autocrineloops may also contribute to growth inhibition and the differentiationprocess in solid tumors such as human melanoma.

Identification of Genes Differentially Expressed During the Processes ofDifferentiation and Growth Suppression in Human Melanoma Cells

To directly identify genes displaying differential expression in humanmelanoma cells induced to terminally differentiate applicants have useda modified subtraction hybridization approach (FIG. 8) (41). cDNAlibraries were prepared from poly (A+) RNA obtained from untreated HO-1cells (Ind⁻ cDNA library; driver cDNA library), and HO-1 cells treatedwith IFN-β+MEZ for 2, 4 8, 12 and 24 (Ind⁺ cDNA library; tester cDNAlibrary). Tester and driver cDNA libraries were directionally clonedinto the commercially available λ Uni-ZAP phage vector. Subtractionhybridization was then performed between double-stranded tester DNA andsingle-stranded driver DNA prepared by mass excision of the libraries.The subtracted cDNAs were efficiently cloned into the λ Uni-ZAP phagevector, which permits easy manipulation for both screening and genecharacterization. A single round of subtraction of untreated HO-1control (Ind⁻) cDNAs from IFN-β+MEZ-treated (Ind⁺) cDNAs resulted in theidentification of a series of cDNAs displaying differential expressionin untreated versus differentiation inducer-treated HO-1 cells. ThesecDNAs are referred to as melanoma differentiation associated (mda)cDNAs. Initially 70 cDNA clones were analyzed and 23 clones were foundto display differences in gene expression between Ind⁻ - and Ind⁺-treated HO-1 cells (41). As anticipated, subtraction of control HO-1cDNAs from IFN-β+MEZ-treated HO-1 cDNAs resulted in a series of MDAgenes that displayed enhanced expression after 24-h treatment with thevarious inducers. These included genes displaying enhanced expression inHO-1 cells treated with both IFN-β and IFN-β+MEZ (mda-1 and mda-2), MEZand IFN-β+MEZ (mda-3), IFN-β, MEZ and IFN-β+MEZ (mda-4) and uniquely byIFN-β+MEZ (mda-5 and mda-6) (FIG. 10) (41). Of these six mda genes, onlymda-3 originally represented a previously identified gene, GOS-19-1(41). Analysis of eight additional human melanoma cell lines indicatesthat specific mda genes also display enhanced expression following a24-h treatment with IFN-β+MEZ (data not shown).

The studies described above indicate that specific mda genes exhibitelevated expression in HO-1 cells after 24-h treatment with appropriateinducing agents. Studies were performed to monitor mda expression inHO-1 cells treated with IFN-β, MEZ or IFN-β+MEZ for 96-h (FIG. 15).Additionally, the pattern of expression of specific mda genes underexperimental conditions inducing reversible growth suppression(IFN-β+IFN-γ, IFN-β, IFN-γ, MEZ, MPA, MPA+MEZ, RA+MEZ), increasedmelanin synthesis (IFN-β, MEZ, MPA, RA, IFN-β+MEZ, MPA+MEZ, RA+MEZ),increased melanin synthesis (IFN-β, MEZ, MPA, RA, IFN-β+MEZ, MPA+MEZ,RA+MEZ), morphological changes (MPA, MEZ, IFN-β+MEZ, MPA+MEZ, RA+MEZ) orterminal cell differentiation (IFN-β+MEZ) was determined. Theseexperiments indicate continuous elevated expression of mda-4, mda-5,mda-6 (p21), mda-7, mda-8 and mda-9 in terminally differentiated HO-1cells (FIG. 15). A differential pattern of expression of the mda geneswas observed in HO-1 cells treated with the various differentiation andgrowth modulating agents. Three of the mda cDNAs, mda-4, mda-5 andmda-8, displayed overlapping induction profiles in HO-1 cells. Thesegenes displayed elevated expression in HO-1 cells treated for 96 hr withIFN-β+MEZ, IFN-γ or IFN-β+IFN-γ (FIG. 15). These cDNAs, which have notbeen previously described in any DNA data base, may correspond to newclasses of cytokine-responsive genes. This possibility is currentlyunder investigation. Expression of mda-7 was increased in HO-1 cellstreated for 96 h with agents inducing growth arrest, including IFN-β,MEZ, MPA, IFN-β+IFN-γ, IFN-β+MEZ, MPA+MEZ, RA+MEZ. The degree ofincrease in mda-7 expression was greatest in HO-1 cells treated withIFN-β+MEZ that also induces terminal cell differentiation. Treatment ofHO-1 cells for 96 hr with RA does not induce growth changes or inducemda-7 expression. Similarly, IFN-γ that only marginally inhibits HO-1growth also does not result in significant mda-7 expression. In the caseof mda-9, increased expression in HO-1 cells was only apparent interminally differentiated cells treated with IFN-β+MEZ (FIG. 15).Further studies are in progress to isolate full-length cDNAs for thevarious novel mda genes and to analyze their expression during theprocesses of growth and differentiation in human melanoma and otherhuman cell types.

The Melanoma Differentiation Associated Gene-6 (mda-6) is theCyclin-Dependent Kinase Inhibitor, p21

Cell-cycle regulation results from the ordered activation of a series ofrelated enzymes referred to as cyclin-dependent kinases (CDKs) (42). Innormal cells, CDKs are predominantly found in multiple quaternarycomplexes, consisting of CDK, a cyclin, proliferating cell nuclearantigen (PCNA) and the p21 protein (43,44). p21 controls CDK activity,thereby affecting cell-cycle control and growth in mammalian cells(43-50). Using human glioblastoma cells containing an induciblewild-type p53 tumor suppressor gene and subtraction hybridization, agene called WAF1 (wild-type p53-activated fragment 1) that encodes anM21,000 protein was identified (49,50). WAF1 is the same p21-encodinggene identified using the two-hybrid system as a potent CDK inhibitor,referred to as CPI1 (Cdk-interacting protein 1) (46). p.21 levels havebeen shown to increase in senescent cells (gene referred to as sdi-1;senescent cell-derived inhibitor) (51) and overexpression of p21inhibits the growth of tumor cells (46,49,51). Treatment of wild-typep53 containing cells with DNA damaging agents results in elevatedwild-type p53 protein and increased p21 levels (51). In this context,p21 may directly contribute to G₁ growth arrest and apoptosis resultingin specific target cells after induction of DNA damage (51). Recentstudies also demonstrate that p21 can: directly inhibit PCNA-dependentDNA replication in the absence of a cyclin/CDK; and inhibit the abilityof PCNA to activate DNA polymerase δ by directly interacting with PCNA(52). These studies indicate that p21 is an important component ofgrowth control, cell-cycle progression, DNA replication and the repairof damaged DNA.

Sequence analysis of mda-6 indicates that it is the CDK inhibitor p21(41) (FIGS. 23A+B; GenBank accession number U09579). The cloning of thisgene from a differentiation-inducer treated human melanoma library,indicates that mda-6 (p21) may contribute to the induction of growtharrest observed in terminally differentiated human melanoma cells. LikeWAF1, mda-6 is also induced in human melanoma cells following DNA damageresulting from treatment with methyl methanesulfonate (53). In humanmelanoma, mda-6 expression is increased during terminal differentiation,rapidly induced by incubation in serum free medium and enhanced in cellsgrown to high saturation densities (53). Several lines of evidenceindicate that the expression of mda-6 inversely correlates with melanomaprogression (53). These include: (A) the presence of higher levels ofmda-6 in actively growing melanocytes and nevi and reduced levels inradial and early vertical growth phase primary melanomas as well asmetastatic human melanomas (53); (B) decreased expression of mda-6 inearly vertical growth phase primary human melanoma cells selected forautonomous or enhanced tumor formation in nude mice (53,54); and (C)increased levels of mda-6 mRNA in metastatic human melanoma cellsdisplaying a loss of metastatic potential resulting followingintroduction of a normal chromosome 6 (53,55). Taken together, theserecent studies indicate that p21 (mda-6/WAF1/CIP1/CAP20/sdi-1) mayfunction as a negative regulator of melanoma growth, progression andmetastasis.

CDK inhibitors, in addition to p21, have also been identified (56-59).These include: a 16-kDa protein, p16^(Ink4) (inhibitor ofcyclin-dependent kinase 4), that specifically inhibits cyclin D/Cdk4(56); a 27-kDa inhibitory protein, p27^(Kip1) (kinase inhibitory protein1), induced in transforming growth factor-β-arrested andcontact-inhibited cells (57,58); and a 28-kDa protein, p₂₈ ^(Ick)(inhibitor of cyclin-dependent kinase), that binds to and inhibits thekinase activity of preformed Cdk/cyclin complexes in human cells (59).It is not presently known if any or all of these CDK inhibitorscontribute to the process of differentiation inducer-mediated growtharrest and terminal cell differentiation in human melanoma.

Summary and Perspectives

It is now possible to reprogram cultured human melanoma cells to anearlier stage in their development by treatment with the appropriateinducing agents. This process, an important component of differentiationtherapy, can result in a rapid loss of proliferative potential andterminal differentiation in these cancer cells. By using the appropriateinducers, it is possible to manipulate specific components of thedifferentiation program in a reversible or irreversible (terminal celldifferentiation) manner. This capability results in a powerful modelsystem permitting the systematic dissection of the roles of specificgenes and biochemical pathways in regulating growth, differentiation andoncogenic potential in human melanoma cells. The combination ofIFN-β+MEZ induces an irreversible loss of growth potential and terminalcell differentiation in human melanoma cells. At comparableconcentrations, IFN-β or MEZ alone induce certain components of thedifferentiation process, but they do not induce an irreversible loss ofgrowth potential or terminal differentiation. The process of terminaldifferentiation in human melanoma cells treated with IFN-β+MEZ involvesspecific biochemical, structural, immunological and gene expressionalterations.

To identify the critical gene expression changes associated with andcontrolling terminal differentiation in human melanoma cells applicantshave used a modified subtraction hybridization protocol. This approachhas resulted in the identification of a series of melanomadifferentiation associated (mda) genes, including both previouslyidentified and novel, that display elevated expression in human melanomacells treated with differentiation and growth suppressing agents. One ofthe mda genes, mda-6, is identical to the cyclin-dependent kinaseinhibitor p21 (also referred to as WAF1, CAP20, CIP1, and sdi-1).Initial studies using a panel of unique mda genes indicate thatincreased expression of specific mda genes' results following treatmentwith agents inducing growth arrest and terminal differentiation as wellas defined classes of DNA damaging and chemotherapeutic agents (FIG.24). Specific mda genes also display differential expression as afunction of human melanoma progression, in normal versus tumor-derivedcells of neuroectodermal origins and in additional cell differentiationmodel systems.

Future studies using the mda genes should prove valuable in defining themolecular determinants mediating growth control, tumor progression,response to chemotherapy and terminal cell differentiation in humanmelanoma and other tumors. The mda genes will also prove useful as partof a simple genetic screen for identifying and monitoring agentsinducing specific DNA damage pathways and for identifying agents capableof inducing terminal differentiation in cancer cells. This informationshould prove important in developing improved therapeutic modalities formetastatic melanoma and for additional human malignancies.

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Sixth Series of Experiments

The multistep carcinogenic process is often characterized by discretechanges in cellular phenotype, including resistance to normal growthinhibitory controls and aberrant patterns of differentiation (Fisher &Rowley, 1991; Knudson, 1993; Hoffman & Liebermann, 1994; Jiang et al.,1994). Treatment of specific cancers with differentiation modulatingagents can result in a suppression of growth and the induction of a moremature differentiated phenotype (Sachs, 1978; Jimenez & Yunis, 1987;Waxman et al., 1988, 1991; Fisher & Rowley, 1991; Lotan, 1993). Themechanism underlying these profound effects on cellular physiology arenot currently known. In the case of human melanoma, the combination ofrecombinant fibroblast interferon (IFN-β) and the anti-leukemic compoundmezerein (MEZ) results in an irreversible loss of proliferative capacityand terminal cell differentiation (Fisher et al., 1985; Jiang et al.,1993). This model system, combined with subtraction hybridization, isbeing used to define the molecular basis of growth control and cancercell differentiation (Jiang & Fisher, 1993; Jiang et al., 1994). Using adifferentiation-induction plus subtraction hybridization approach, aseries of differentially expressed cDNAs, termed melanomadifferentiation associated (mda) genes, have been identified thatdisplay enhanced expression as a function of growth suppression andterminal cell differentiation (Jiang & Fisher, 1993; Jiang et al.,1994).

The human p21 cyclin-dependent kinase (Cdk)-interacting protein CIP1(Xiong et al., 1993b); Harper et al., 1993), and the mouse CAP20homologue (Gu et al., 1993), is a ubiquitous inhibitor of cyclin kinasesand an integral component of cell cycle control. This gene is identicalto the WAF1 (wild-type (wt) p53 activated factor-1) gene identifiedfollowing induction by wt p53 protein expression in a human glioblastomamultiforme cell line (El-Deiry et al., 1993). p21 has also beenindependently cloned as a consequence of induction of senescence innormal human foreskin fibroblast cells, SDI1 (senescent cell-derivedinhibitor-1) (Noda et al., 1994), and during the process of terminalcell differentiation in human melanoma cells, mda-6 (Jiang and Fisher,1993; Jiang et al., 1994). p21 is a nuclear localized protein that isinducted by DNA damage and during apoptosis in specific cell types as afunction of wt p53 activation (El-Deiry et al., 1993, 1994). Thesestudies suggest that p21 may be an important downstream mediator of wtp53-induced growth control in mammalian cells (El-Deiry et al., 1993,1994). Somewhat paradoxical data indicates that WAF1/CIP1 is induced asan immediate-early gene following mitogenic stimulation of growtharrested cells in a p53-independent manner (Michieli et al., 1994).Applicants presently demonstrate that mda-6 (WAF1/CIP1/SDI1) expressionis also induced by mechanistically diverse acting agents resulting inmacrophage/monocyte (TPA and Vit D3) or granulocyte (RA and DMSO)differentiation in human promyelocytic leukemia cells (Collins, 1987),HL-60, that lack endogenous p53 genes (Wolf and Rotter, 1985). Usingdifferentiation-resistant variants (Homma et al., 1986; Mitchell et al.,1986), a direct correlation is found between the early induction ofmda-6 expression and the onset of specific programs of differentiationin HL-60 cells. Applicants' results indicate that sustained p21expression can be maintained in the absence of wt p53 protein andelevated levels of p21 (WAF1/CIP1/SDI1) mRNA and protein correlate withgrowth suppression and differentiation induction in a p53-independentmanner in HL-60 cells.

Experimental Results

Treatment with Diverse Acting Differentiation Inducing Agents Results inIncreased mda-6 (WAF1/CIP1/SDI1) Expression in HL-60 Cells

HL-60 is a differentiation competent myeloid leukemia cell line that canbe induced to differentiate along both monocytic and granulocyticlineages following exposure to the appropriate inducing agents(Gallagher et al., 1979; Huberman & Callaham, 1979; Breitman et al.,1980; Collins 1987). Treatment of HL-60 cells with TPA, that committhese cells to a macrophage-like lineage (Lotem & Sachs, 1979; Rovera etal., 1979), induces mda-6 expression as detected by both Northernblotting (FIGS. 25A-B) and RT-PCR (FIGS. 26A-C). Induced expressionfollowing TPA treatment (3 nM) occurs within 2 h of exposure andelevated mda-6 levels persist in terminally differentiated HL-60 cells(FIGS. 25A-B and 27A-C). Similarly, Vit D3 (400 nM), that also commitsHL-60 cells to a monocyte-macrophage-like lineage (Miyaura et al., 1981;Tanaka et al., 1982), induces mda-6 within 1 h of treatment and elevatedexpression continues in terminally differentiated HL-60 cells (FIGS.26A-C and 27A-C). Induction of a granulocyte-like phenotype in HL-60cells by RA (Breitman et al., 1980) or DMSO (Collins et al., 1978) alsoinduces mda-6 mRNA production. In the case of RA (1 μM), induction ofmda-6 is apparent within 3 h and expression remains elevated 6 dayspost-RA treatment (FIGS. 25A-B, 26A-C and 27A-C). DMSO (1%) also inducesmda-6 by 3 h and augmented expression persists at day 5 when cells areterminally differentiated (data not shown). These results indicate thatinduction of both macrophage/monocyte and granulocyte differentiationpathways in human myeloid leukemia HL-60 cells results in the inductionof mda-6 expression. In addition, mda-6 expression is induced during theearly commitment stage of HL-60 differentiation and persists interminally differentiated cells.

To determine if the elevation in mda-6 expression with an increase inMDA-6 (WAF1/CIP1/SDI1) protein, HL-60 cells were labeled for 4 h with ³⁵S-methionine after 12, 24, 48 and 72 h treatment with TPA (3 nM), DMSO(1%) or RA (1 μM) and lysates were immunoprecipitated using WAF1/CIP1antibody (FIG. 28). As a control for protein loading, the level of ACTINprotein was determined by immunoprecipitation. Although no MDA-6 proteinwas detected in HL-60 cells, 12 h treatment with TPA or RA resulted inimmunologically reactive MDA-6 protein. In 1% DMSO treated HL-60, MDA-6protein was first apparent by 48 h. The levels of MDA-6 proteinincreased in a temporal manner with all three inducers and the highestlevels were apparent at 72 h. The most active inducer of MDA-6 protein,as well as the most active growth suppressing agent, was TPA. Polyclonalantibodies prepared against N-terminal peptide regions of MDA-6 alsoimmunoprecipitated MDA-6 from differentiation inducer treated HL-60cells and human melanoma cells. In contrast, using a monoclonal antibody(PAb 421) that reacts with both wild-type and mutant p53, no reactiveprotein was detected after immunoprecipitation of ³⁵ S-methioninelabeled lysates prepared from HL-60 cells and TPA-, DMSO- or RA-treatedHL-60 cells (data not shown). These results provide direct evidence thatinduction of elevated mda-6 mRNA expression in differentiationinducer-treated HL-60 cells results in elevated MDA-6 protein levels inthe absence of p53 protein.

Induction of mda-6 Expression is Altered in Differentiation-resistantHL-60 Variants

The availability of variants of HL-60 cells displaying resistance toTPA-induced growth suppression and differentiation (Murao et al., 1983;Fisher et al., 1984; Anderson et al., 1985; Mitchell et al., 1986; Hommaet al., 1986, 1988; Tonetti et al., 1992) provides a valuableexperimental model to evaluate the potential involvement of mda-6 inthese processes. By continuously growing HL-60 cells in graduallyincreasing concentrations of TPA (up to 3 μM), the TPA-resistant HL-60variant HL-525 was developed (Homma et al., 1986; Mitchell et al.,1986). These cells were used to determine the kinetics of mda-6expression as a function of short term (1 through 12 h) and long-term (1through 6 d) treatment with TPA, Vit D3 and RA. As anticipated, HL-525cells demonstrate a suppressed response to mda-6 induction following TPAtreatment (FIGS. 26A-C). Parental HL-60 cells treated with 3 nM TPA showmda-6 expression within 2 h, whereas induction in HL-525 cells is notapparent until 12 h treatment (FIGS. 26A-C and 27A-C). Vit D3 (400 nm)treatment of HL-60 parental cells results in mda-6 expression after 1 hwith a continued increase over the 12 h test period (FIGS. 26A-C). InHL-525 cells, mda-6 expression is observed by 2 h following Vit D3treatment and the level of expression in the variant cells after 12 hexposure to 400 nM Vit D3 is lower than seen in HL-60 cells similarlytreated for 1 h. RA (1 μM) induces mda-6 expression after 3 h in HL-60cells and the level of mda-6 expression increases over the 12 h testperiod. In contrast, no induction of mda-6 is apparent in RA-treatedHL-525 cells by 12 h. These results indicate that the TPA-resistantHL-525 cells differ from HL-60 cells with respect to the early inductionof mda-6. This defective early induction of mda-6 in HL-525 cells ismost evident following RA or TPA treatment, whereas Vit D3 results in areduced capacity to induce mda-6 that is less dramatic than with theother inducers. Since these studies involve separate RT-PCR reactions,direct quantitation is difficult, however, it appears that both theearly kinetics of induction and the final level of induction of mda-6are reduced in HL-525 cells in comparison with HL-60 parental cells.These observations are supported by Northern blotting analyses ofsimilar samples (data not shown).

RT-PCR analyses were used to determine the effect of extended treatmenttimes (12 h to 6 d) on induction of mda-6 in HL-60 and HL-525 cells(FIGS. 27A-C). With the three inducers, mda-6 expression in HL-60 cellswas apparent during all of the extended treatment times. In the case ofHL-525, mda-6 expression was also induced at all of the time points byTPA and Vit D3. On the basis of direct quantitation and an adjustmentfor amplification of GAPDH, the levels of mda-6 induction in TPA- andVit D3-treated HL-525 cells appear to be lower than in similarly treatedHL-60 cells (FIGS. 27A-C). However, since RT-PCR assay employed is notquantitative (no internal GAPDH control was used in the sameamplification reaction), further studies using quantitative RT-PCR andNorthern blotting will be required to verify this conclusion. In thecase of RA treated HL-525 variant cells, a delay in induction of mda-6is evident, i.e. expression is not detected until 2 d post-treatment(FIGS. 27A-C). These results suggest that the HL-525 TPA-resistantvariant may also display an attenuated response to mda-6 expressionfollowing extended treatments with RA and Vit D3 as well as TPA.

Previous studies indicate that a 6 d growth of HL-60 cells is stronglyinhibited (60 to 98% reduction in cell number relative to control) by 3nM TPA, 1 μM RA and 400 nM Vit D3 with TPA being the most effective ofthe group (Murao et al., 1983 and data not shown). In the HL-525variant, only a slight reduction (less than 5%) in growth is observedafter treatment with TPA or Vit D3. Treatment with 1 μM RA for 6 dresults in a 35% reduction in cell numbers. These results indicate thatthe TPA-resistant variant displays some cross-resistance to the growthsuppressive effects of RA and it is relatively refractive to the dose ofVit D3 used for the present studies. Analysis of OKM1 reactivity, whichreacts with human blood monocytes and granulocytes (Foon et al., 1982),in HL-60 indicates that both TPA and Vit D3 are similarly active, whileRA displays a somewhat reduced activity (Table 4). In HL-60 cells, anincrease in OKM1 positive cells is seen with all of the inducers overtime, with maximum induction observed at 6 d with TPA and Vit D3 and by4 d with RA. In contrast, HL-525 cells treated with TPA do not display asignificant increase in OKM1 positive cells, whereas RA induces a smalleffect at 6 d and Vit D3 is an effective inducer of OKM1 reactivity atboth 4 and 6 d. These observations indicate that the effects of Vit D3on growth suppression and induction of differentiation are not directlycorrelated processes in HL-525 cells, whereas growth suppression andinduction of differentiation are related changes in Vit D3 treated HL-60parental cells.

                  TABLE 4                                                         ______________________________________                                        OKM1 reactivity in HL-60 and HL-525 cells treated with                          differentiation inducers                                                                     Days of induction                                            Cell type   Inducer  2          4   6                                         ______________________________________                                        HL-60                                                                            Untreated  3 5 12                                                             TPA 55 81 95                                                                  RA 49 76 77                                                                   Vit D3 -- 88 95                                                              HL-525                                                                         Untreated  5 3 5                                                              TPA -- 8 4                                                                    RA -- 10 13                                                                   Vit D3 -- 84 80                                                            ______________________________________                                         Cells were seeded into 100mm Petri dishes at 1.5 × 10.sup.5 cells/m     and TPA (3 nM), RA (1 μM) or Vit D3 (400 nM) added 12 h later. OKM1        reactivity was assessed at the indicated times as described in Materials      and methods. Results are expressed as percent OKMI positive cells.            - = Not determined.                                                      

mda-6 is an Immediate-early Gene Induced in HL-60 Cells in the Presenceof Cycloheximide

To determine if the induction of mda-6 expression requires continuousprotein synthesis, the effect of the protein synthesis inhibitorcycloheximide (CHX) on de novo and TPA-induced mda-6 expression wasdetermined (FIGS. 29A-B). Treatment of HL-60 cells with CHX for 1, 3, 6or 10 h results in the induction of mda-6. In addition, the relativelevel of mda-6 induction is unaffected when HL-60 cells aresimultaneously treated with TPA and CHX. In HL-525 cells, CHX alsoinduces mda-6 expression, but the level of induction is lower than inHL-60 parental cells. In contrast, when HL-525 cells are treated withboth CHX and TPA, mda-6 expression is increase (superinduction) to agreater extent than with CHX alone (FIGS. 29A-B). These resultsindicated that mda-6 is an immediate-early response gene in HL-60 cellsand induction does not require ongoing protein synthesis. The ability ofCHX alone to induce mda-6 expression in HL-60 and to a lesser extent inHL-525 cells suggests that mda-6 expression may be controlled by anunstable suppressor. In the case of HL-525, inhibition in TPA-induceddifferentiation may be related to alterations in the levels of thisunstable suppressor.

Experimental Discussion

Terminal differentiation in diverse cell types occurring eitherspontaneously or as a consequence of treatment with specific inducingagents correlates with an irreversible loss of proliferative potential(Sachs, 1978; Jimenez & Yunis, 1987; Waxman et al., 1988; 1991; Fisher &Rowley, 1991; Hoffman & Liebermann, 1994). The specific gene expressionchanges and proteins that mediate growth arrest and induction ofdifferentiation in the majority of differentiation models remain to bedefined. Using subtraction hybridization and agents capable of inducingterminal cell differentiation in human melanoma cells, mda genes havebeen identified whose expression directly correlate with growth arrestand terminal cell differentiation (Jiang & Fisher, 1993; Jiang et al.,1994). One such gene, mda-6 is identical to WAF1/CIP1/CAP20/SDI1 thatencodes the ubiquitous inhibitor of cyclin-dependent kinases, p21 (Jiang& Fisher, 1993; Jiang et al., 1994). A direct effect of p21 on growthhas been demonstrated by transfecting expression constructs intomammalian cells (El-Deiry et al., 1993; Harper et al., 1993; Jiang etal., in preparation). Although induction of p21 expression was initiallyconsidered to be dependent upon wild-type p53 protein (El-Deiry et al.,1993, 1994), recent studies suggest that this assumption must bereevaluated (Michieli et al., 1994). These include the ability ofmitogens to transiently stimulate p21 expression in quiescentfibroblasts from p53 knock out mice lacking p53 protein (Michieli etal., 1994) and the decreased expression of p53 mRNA and protein but theincreased expression of mda-6 mRNA and protein in terminallydifferentiated human melanoma cells (Jiang et al., in preparation). Inthe present study, definitive evidence is presented that p21(mda-6/WAF1/CIP1/CAP20/SDI1) is an immediate-early response gene that isinduced in the absence of p53 protein as a function of growth arrest andinduction of differentiation in HL-60 cells. The ability of diverseinducers, including TPA and Vit D3 that produce monocyte and macrophagedifferentiation and RA and DMSO that elicit granulocyte differentiation,to stimulate p21 mRNA synthesis and p21 protein in HL-60 cells definesthis early genotypic change as an important component of growth arrestand terminal differentiation in myeloid leukemic cells.

Previous studies have demonstrated that TPA-resistant variants of HL-60cells, isolated in the absence of mutagenesis, display a number ofbiochemical and cellular traits that distinguish them from parentalTPA-sensitive HL-60 cells (Murao et al., 1983; Fisher et al., 1984;Anderson et al., 1985; Homma et al., 1988; Tonetti et al., 1992).TPA-resistant HL-60 variant cells, such as HL-525, display alteredprotein kinase C (PKC) isozyme profiles, including the absence of PKC-βand possibly a δ-like PKC gene expression (Tonetti et al., 1992). TPAresistance in HL-60 cells is associated with decreased fluidity ofeither the inner leaflet of the plasma membrane and/or of the cytosolicorganellar membranes (Fisher et al., 1984). A striking biochemicaldifference between HL-60 and TPA-resistant HL-60 variants is theinability of the latter cell type to translocate PKC from the cytosol tothe membrane fraction following TPA treatment (Homma et al., 1986).TPA-resistant HL-60 variants also display modifications in proteinphosphorylation patterns after TPA treatment (Homma et al., 1988) andaltered responses in immediate-early gene expression following TPAtreatment (Tonetti et al., 1992). Of most direct relevance to thepresent study is the observation by Tonetti et al. (1992) that theHL-525 variant displays an attenuated response to TPA induction ofimmediate-early genes, including c-fos, c-jun and jun-B. The level ofc-fos and jun-B induction is substantially greater in TPA-treated HL-60parental cells and in the TPA-sensitive HL-60 clone, HL-205, than in twoTPA-resistant clones, HL-525 and HL-534. In the case of c-jun, TPA failsto induce this gene expression change in HL-525 or HL-534 cells, whereasinduction is apparent in HL-60 and HL-205 cells by 9 h post-treatmentwith TPA (Tonetti et al., 1992).

In the present study, applicants demonstrate that the TPA-resistantvariant HL-525 displays a diminished response to TPA-induction of mda-6in comparison with HL-60 cells (FIGS. 26A-C and 27A-C). The HL-525 cellslikewise differ from HL-60 cells in the induction of mda-6 followingtreatment with Vit D3 and RA (FIGS. 26A-C and 27A-C). With both of theseagents, the temporal pattern of induction and the magnitude of inductionof mda-6 are diminished in HL-525 cells. The TPA-resistant HL-525variant also displays a reduced susceptibility to growth arrestfollowing treatment with Vit D3 and RA, whereas Vit D3 still has thecapacity to induce differentiation as monitored by OKT1 reactivity(Table 4). These findings indicate that these two phenomena, i.e.,growth arrest and differentiation, are dissociable processes in HL-525cells. Unlike TPA- and RA-treated HL-525 cells, Vit D3 treated cellsshow early induction of mda-6 (after 2 h treatment) (FIGS. 26A-C).Although further studies are required, it is tempting to speculate thatthe early induction of mda-6 by Vit D3 may be a primary determinantcommitting HL-525 cells to differentiate, whereas the absence ofsufficient accumulated levels of p21 protein, encoded by mda-6, inlong-term treated cultures (6 d) precludes growth arrest.

CHX induces mda-6 in HL-60 cells and the ability of TPA to induce mda-6expression is not inhibited by CHX indicating that mda-6 is animmediate-early response gene. In HL-525 cells, CHX induces mda-6 lesseffectively than in HL-60 cells, whereas the combination of CHX and TPAresults in a superinduction of mda-6 in this TPA-resistant variant(FIGS. 29A-B). These observations and previous investigations supportthe hypothesis that the HL-525 variant cells may be defective in signaltransduction processes, possibly involving PKC-β and a δ-like PKC gene,that prevent or reduce the induction of immediate-early response genes,including c-fos, c-jun, jun-B and p21. The lower levels of theimmediate-early response genes following TPA treatment may then impedethe induction of subsequent cellular genes involved in the initiation ofterminal differentiation in HL-525 cells. Induction of theimmediate-early gene p21 during the commitment phase of HL-60differentiation may be an important component in initiatingdifferentiation, whereas a continued elevation of p21 may be requiredfor growth arrest and maintenance of terminal differentiation in HL-60cells. In the case of the HL-525 variant, treatment with TPA does notinduce the early induction of p21 and it may not generate sufficientlyhigh levels of p21 to produce sustained growth arrest.

Recent studies are providing new insights into the mode of action ofp21. The p21 protein was originally identified as part of quaternarycyclin D complexes in human diploid fibroblasts, that also possesscyclin-dependent kinases (CDK) and proliferating cell nuclear antigen(PCNA) (Xiong et al., 1992). Subsequent studies demonstrated that p21and PCNA can form multiple quaternary complexes with all cyclins andCDKs in normal human fibroblasts, but not in virally transformed cells(Xiong et al., 1993a). p21 has also been shown to associate with andinhibit the activity of all cyclin-CDK enzymes (Xiong et al., 1993b;Harper et al., 1993; Gu et al., 1993). Recent experiments demonstratethat p21 can directly complex with and inhibit PCNA suggesting that thisprotein may be a critical regulator of DNA replication, DNA repair andcell cycle machinery (Waga et al., 1994). The importance of p21 in cellcycle and growth control has been reinforced by the independentisolation of this gene by virtue of its induction by the tumorsuppressor p53 [WAF1, (El-Deiry et al., 1993)], as a direct regulator ofCDK2 using the two-hybrid screening technique [CIP1, Harper et al.,1993), as a cDNA from senescent cells with the ability to inhibit theability of young cells to enter S phase after overexpression followingtransient transfection [SDI1, (Noda et al., 1994)] and as adifferentially expressed differentiation related cDNA isolated from ahuman melanoma cell library using subtraction hybridization (mda-6,Jiang & Fisher, 1993; Jiang et al., 1994). The level of p21 has beenshown to vary depending on the specific stage of the cell cycle (Li etal., 1994). In IMR90 normal diploid fibroblast cells released from serumstarvation, the levels of p21 are maximum immediately after serumstimulation, start to decrease as cells reach the G1/S boundary, displaylowest levels during S phase, and increase again as cells leave the Sphase and enter the G2 and M phase (Li et al., 1994). These observationsindicate that p21 may contribute to both the G1/S and the G2/Mcheckpoint pathways. The interaction of p21 with cyclin and CDK duringthe cell cycle is not random, but rather occurs when the specificcyclin-CDK enzyme is reputed to function (Li et al., 1994). Moreover,the increased level of p21 in quiescent and terminally differentiatedcells suggests that this protein may play a crucial role in preventingthese cells from re-entering the cell cycle, an absolute requirement forterminal differentiation.

In summary, the ability of different inducers of growth suppression andterminal differentiation to induce p21 early in the differentiationprocess and the persistence of elevated levels of p21 after terminaldifferentiation in the p53 negative HL-60 cell line indicates animportant role for this inhibitor of cyclin-dependent kinases indifferentiation. Further support for an involvement of p21 in growthcontrol and differentiation is indicated by the ability of structurallydiverse inducers of differentiation to induce p21 expression and growtharrest in additional cell lines, including human melanomas (Jiang &Fisher, 1993; Jiang et al., 1994) and human neuroblastomas (Jiang etal., in preparation). Moreover, the diminished ability of specificinducers of differentiation to produce growth arrest and differentiationin the TPA-resistant HL-525 variant also correlates with a reduced earlyand sustained induction of p21. Further studies to determine if forcedexpression of p21, using inducible expression vectors, are sufficient toinduce an irreversible loss in proliferative capacity and terminaldifferentiation in HL-60 cells, and other differentiation competent cellculture systems, appears warranted and are currently in progress. Theseexperiments will permit a direct functional evaluation of p21 inregulating both cellular growth and differentiation in the absence andpresence of wild-type p53.

Materials and Methods

Cells and Culture Conditions

HL-60 cells were originally provided by Dr. R. C. Gallo (National CancerInstitute, Bethesda, Md.) (Collins et al., 1978; Huberman & Callaham,1979). HL-60 cells designated HL-525 were derived by cloning HL-60 cellsafter subculturing 102 times in the presence of increasingconcentrations (up to 3 μM) of TPA at 5- to 8-day intervals (Homma etal., 1986; Mitchell et al., 1986). The HL-525 cell variant displays astable phenotype with regards to resistance to induction of celldifferentiation by TPA for at least 50 to 60 subcultures (200 to 300cell generations). Prior to the experiments described in this study, theHL-525 cells were subcultured more than 20 times in the absence of TPA.Cells were grown in 100-mm tissue culture dishes in RPMI 1640 mediumsupplemented with 20% fetal bovine serum, penicillin (100 units/ml) andstreptomycin (100 μg/ml) (Grand Island Biological Co., NY) at 37° C. inan atmosphere of 5% CO₂ in air in a humidified incubator. The 1,25-(OH)₂D₃ (Vit D3) and TPA were dissolved in a final concentration of 0.01%DMSO, and all-trans retinoic acid (RA) was dissolved in 0.1% DMSO inculture medium. DMSO at these concentrations did not affect cell growthor the expression of the various differentiation markers. Controlcultures were treated with DMSO at a final concentration of 0.1% inculture medium. For experiments designed to test the effect of DMSO onHL-60 and HL-525 cells, DMSO was added to tissue culture medium at afinal concentration of 1%. Stock solutions of CHX (10 mg/ml) wereprepared in culture medium. CHX was added at a final concentration of 10μg/ml. To examine a requirement for protein synthesis on mda-6 geneexpression, HL-60 or HL-525 cells were seeded into 150 mm Petri dishes(5×10⁵ cells/ml) in 30 ml of medium. CHX was added to a finalconcentration of 10 μg/ml 15 min prior to the addition of TPA to 3 nM.Cells were harvested at various times after addition of TPA forsubsequent RNA isolation and analysis.

Measurement of Differentiation and Growth

Cell counts were determined by hemocytometer chamber counting.Immunofluorescence tests for reactivity with the OKM1 antibody (OrthoPharmaceutical Corp., Raritan, N.J.) were performed as previouslydescribed (Murao et al., 1983).

RNA Isolation, Northern Blotting and RT-PCR

RNA was purified by centrifugation through a CsC1 cushion as describedby Chirgwin et al. (1979). Ten μg of RNA was denatured withglyoxal/DMSO, electrophoresed on 1.0% agarose gels, transferred to nylonmembranes, hybridized to a ³² P-labeled mda-6 probe (Jiang and Fisher,1993) and then after stripping the membrane hybridized to a 32P-labeledrat GAPDH probe (Fort et al., 1985), as described previously (Reddy etat., 1991; Su et al., 1991; Jiang et al., 1992). Followinghybridization, the filters were washed and exposed for autoradiography(Reddy et al., 1991; Su et al., 1991; Jiang et al., 1992). mda-6 andGAPDH gene expression were also determined by reversetranscription-polymerase chain reaction (RT-PCR) as described (Adollahiet al. 1991; Lin et al., 1994). Total cytoplasmic RNA was treated with0.5 units DNase (Boehringer-Mannheim Biochemicals)/μg RNA in 15%glycerol, 10 mM Tris, pH 7.5, 2.5 mM MgCl₂, 0.1 mM EDTA, 80 mM KCl, 1 mMCaCl₂ and 1 unit/ml RNasin (Promega) at 30° C. for 10 min. RNA wasextracted with phenol-chloroform, precipitated with sodiumacetate/ethanol and RNA pellets were resuspended indiethylpyrocarbonate-treated H₂ O. One μg of total RNA was reversetranscribed with 200 units of murine leukemia virus reversetranscriptase (Bethesda Research Laboratories) in 20 μl containing 1 mMdeoxyribonucleotide triphosphates, 4 mM MgCl₂, 10 mM Tris, pH 8.3, 50 mMKCl, 0.001% gelatin and 0.2 μg oligo-dT primer. Samples were diluted to100 μl with buffer containing 0.2 mM deoxyribonucleotide triphosphates,2 mM MgCl₂, 10 mM Tris, pH 8.3, 50 mM KCl and 0.001% gelatin. Fifty pmolof each primer, 1.5 units Taq DNA polymerase Perkin-Elmer Cetus) wereadded and samples were covered with mineral oil, heated at 95° C. for 5min and subjected to 20 cycles of PCR in a Perkin-Elmer Thermal Cyclerusing 2 min denaturation at 95° C., 1 min annealing at 55° C. and 4 minpolymerization at 72° C. After extraction with chloroform, 20 μg ofproducts were electrophoresed, blotted onto nylon filters and hybridizedwith an mda-6 or GAPDH specific probe. The template primers for mda-6were 5' to 3' CTCCAAGTACACTAAGCACT (SEQ ID NO:18) andTAGTTCTACCTCAGGCAGCT (SEQ ID NO:19) (GenBank accession number U09579)and the template primers for human GAPDH were 5' to 3°CATGGCCTCCAAGGAGTAAGA (SEQ ID NO:20) and CGTCTTCACCACCATGGAGAA (SEQ IDNO:21) (GenBank accession number J02642).

Immunoprecipitation Analyses

Immunoprecipitation analyses were performed as described previously(Duigou et al., 1991; Su et al. 1993). Logarithmically growing HL-60cells were either untreated or treated for 12, 24, 48 or 72 h with TPA(3 mM), RA (1 μM) or DMSO (1%) in 10-cm plates. Cultures were starved ofmethionine for 1 h at 37° C. in methionine-free medium, cells wereconcentrated by pelleting and labeled for 4 h at 37° C. in 1 ml of thesame medium with 100 μCi of [₃₅ S] (NEN; Express ₃₅ S). After labeling,the cells were washed twice with ice-cold phosphate-buffered saline andlysed for 1 h on ice by the addition of RIPC (20 mM Tris-base, pH 7.5,500 mM NaCl, 0.05% Nonidet P-40, 100 μg/ml phenylmethylsulfonyl fluorideand 0.02% sodium azide). The lysate was clarified by centrifugation inan Eppendorf microfuge at 10,000×g for 10 min at 4° C. Samplescontaining 4×10⁶ counts were incubated with 2 μg of WAF1/CIP1 (C-19)(Santa Cruz Biotechnology) (or MDA-6 peptide-derived) rabbit polyclonalIgG or actin monoclonal antibody (Oncogene Sciences) with rocking at 4°C. for 24 h. The next day, 30 μl (packed volume) of protein G-agarose(Oncogene Sciences) was added to each tube, and incubation with rockingat 4° C. continued for another hour. The protein G pellets were thenwashed five times with 1 ml of ice-cold RIPC:phosphate-buffered saline(1:1, v/v). Thirty μl of sodium dodecyl sulfate-polyacrylamide gelelectrophoresis buffer were added to the pellets, and the sample washeated at 87° C. for 3 min. The samples were loaded onto an 10%polyacrylamide gel and run overnight at 40 V. The gel contained Rainbowprotein markers (Amersham) for sizing. Gels were fixed with 10% aceticacid plus 10% methanol for 30 min, incubated in DMSO for 30 min,incubated with 10% 2,5-diphenyloxazole in DMSO for 30 min, washed threetimes with cold water (10 min each), dried and exposed to film.

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Seventh Series of Experiments

The combination of recombinant human fibroblast interferon (IFN-β) andthe antileukemic compound mezerein (MEZ) induces terminaldifferentiation with an irreversible loss of proliferative capacity inhuman melanoma cells. Using subtraction hybridization, cDNAs wereidentified that display enhanced expression in terminally differentiatedand growth arrested human melanoma cells (Jiang and Fisher, 1993; Jianget al., 1994). A specific melanoma differentiation-associated (mda)cDNA, mda-6, is described whose expression inversely correlates withmelanoma progression and growth. mda-6 is identical to WAF1/CIP1/SDI1that encodes the M_(r) 21,000 protein (p21) that is an inhibitor ofcyclin-dependent kinases. Actively growing normal melanocyte,SV40-immortalized human melanocyte and dysplastic nevus cell linessynthesize elevated levels of mda-6 mRNA; whereas, activelyproliferating radial and early vertical growth phase primary melanomasas well as metastatic human melanoma cells produce reduced levels ofmda-6 mRNA. Treatment of primary and metastatic human melanoma cellswith IFN-β+MEZ results in growth inhibition and an increase in mda-6expression. mda-6 expression also increases when human melanoma cellsare grown to high saturation densities or when grown in serum-freemedium. Using anti-p53 and anti-p21 antibodies, an inverse correlationis found between p53 and p21 protein levels during growth arrest anddifferentiation. Induction of growth arrest and terminal differentiationin HO-1 human melanoma cells by IFN-+MEZ results in a temporal decreasein wild-type p53 protein levels with a corresponding increase in p21levels. In the Matrigel-assisted melanoma progression model, mda-6expression decreases in early vertical growth phase primary humanmelanoma cells selected for autonomous or enhanced tumor formation innude mice. In metastatic human melanoma cells displaying a loss ofmetastatic potential resulting following introduction of a normal humanchromosome 6, mda-6 mRNA levels increase. Taken together, these studiesindicate that mda-6 (p21) may function as a negative regulator ofmelanoma growth, progression and metastasis.

Development of malignant melanoma in humans, with the exception ofnodular type melanoma, often consists of a series of sequentialalterations in the evolving tumor cells (for reviews see: Kerbel, 1990;Herlyn, 1990; Clark, 1991). These changes may include conversion of anormal melanocyte into a common acquired melanocytic nevus (mole),followed by the development of a dysplastic nevus, a radial growth phase(RGP) primary melanoma, a vertical growth phase (VGP) melanoma andultimately a metastatic melanoma. Melanomas are readily treatable duringthe early stages of development; however currently employed therapiesare not very effective (<20% survival) in preventing metastatic spreadand morbidity in patients with VGP lesions >4.0-mm thickness. Theseobservations indicate an imperative for improved therapeutic modalitiesfor treating patients with this malignancy.

A potentially less toxic strategy for cancer therapy involves aprocedure termed "differentiation therapy" (Sachs, 1978; Jimenez &Yunis, 1987; Waxman et al., 1988, 1991; Fisher & Rowley, 1991; Lotan,1993; Jiang et al., 1994a). An essential premise underlying thisapproach is that specific cancers have reversible defects in normalprograms of differentiation and growth control. By using specific singleor combinations of agents, it has been possible to slow or stopproliferation of cancer cells and correspondingly increase expression ofdifferentiation-associated properties (for reviews see: Waxman et al.,1988, 1991; Fisher & Rowley, 1991; Lotan, 1993; Jiang et al., 1994a).

Treatment of human melanoma cells with IFN-β+MEZ results in rapid,irreversible loss of proliferative capacity, an induction of specificchanges in gene expression, a modification in cell surface antigenicprofile and terminal cell differentiation (Fisher et al., 1985, 1986;Guarini et al., 1989, 1992; Graham et al., 1991; Jiang & Fisher, 1993;Jiang et al., 1993, 1994a). The ability of IFN-β+MEZ to induce terminaldifferentiation in human melanoma cells is hypothesized to be aconsequence of activation by these agents of genes that negativelycontrol melanoma growth and induce differentiation. Two predictionsarise from this model: specific genes involved in growth control anddifferentiation in human melanoma cells are expressed at diminishedlevels in melanoma versus their normal melanocyte counterpart; and thecombination of IFN-β+MEZ can induce enhanced expression of specificmelanoma differentiation associated (mda) genes, growth suppression andterminal cell differentiation (Jiang & Fisher, 1993; Jiang et al.,1994a). To test these possibilities and to directly identify and clonegenes that would fulfill this scheme, a modified subtractionhybridization approach was used that employed the human melanoma cellline HO-1 treated with IFN-β+MEZ (Jiang & Fisher, 1993). Using thisstrategy a series of melanoma differentiation associated (mda) geneswere cloned that display enhanced expression in HO-1 human melanomacells treated with IFN-β+MEZ (Jiang & Fisher, 1993; Jiang et al.,1994a). mda-6 encodes the cyclin dependent kinase inhibitor protein, p21(Jiang et al., 1994a, 1994b). p21 has been cloned by a number oflaboratories by virtue of its ability to interact with and inhibitcyclin dependent kinases (CIP1: cyclin-dependent kinase(CDK)-interacting protein-1) (Harper et al., 1993), its induction bywild-type p53 (WAF1; wild-type (wt) p53 activated factor-1) (El-Deiry etal., 1993), its induction during senescence (SDI1; senescentcell-derived inhibitor-1) (Noda et al., 1994) and its induction as afunction of growth arrest and terminal differentiation in human melanomacells (mda-6; melanoma differentiation associated gene-6) (Jiang &Fisher, 1993; Jiang et al., 1994a). Although originally assumed to bedependent on wt p53 for induction (El-Deiry et al., 1993, 1994), recentstudies indicate that p21 can also be induced in a p53-independentmanner (Michieli et al., 1994; Jiang et al., 1994b; Steinman et al.,1994). In the present study, applicants demonstrate that the levels ofp21 protein increase as the levels of wt p53 protein decrease duringIFN-β+MEZ induction of growth suppression and terminal differentiationin HO-1 human melanoma cells. These results reveal a novel associationbetween p21 and p53 in the process of melanoma growth anddifferentiation. Moreover, applicants show a relationship betweenexpression of mda-6 and melanoma evolution, differentiation and growth.These results provide a direct link between alterations in p21expression and cancer progression.

Experimental Results

Increased mda-6 Expression in Human Melanoma Cells During GrowthInhibition and Terminal Cell Differentiation

To define the molecular basis by which IFN-β+MEZ induces irreversiblegrowth arrest and terminal differentiation in human melanoma cellsapplicants used the technique of subtraction hybridization withuninduced and differentiation inducer treated HO-1 human melanoma cDNAlibraries (Jiang & Fisher, 1993). Using this strategy, an mda-6 cDNA wasidentified in a differentiation inducer (IFN-β+MEZ) treated subtractedHO-1 human melanoma library that displays differential expression as afunction of IFN-β+MEZ induced growth arrest and terminal differentiation(Jiang & Fisher, 1993; Jiang et al., 1994a). By screening adifferentiation inducer-treated HO-1 cDNA library (Jiang & Fisher, 1993)and using rapid amplification of cDNA ends (RACE) (Frohman et al., 1988;Loh et al., 1989; Ohara et al., 1989) a full-length mda-6 cDNA wascloned (Jiang et al., 1994a). mda-6 contains the same open reading frameas WAF1 (El-Deiry et al., 1993), CIP1 (Harper et al., 1993), CAP20 (Guet al., 1993) and SDI1 (Noda et al., 1994) (FIG. 30). These genes encodethe ubiquitous inhibitor of cyclin dependent kinases, p21.

Treatment of actively growing HO-1 cells for 24 h with IFN-β+MEZ resultsin increased mda-6 mRNA levels and growth inhibition (Fisher et al.,1985; Jiang & Fisher, 1993; Jiang et al., 1993) (FIGS. 31A-E). Incontrast, treatment of HO-1 cells with either IFN-β or MEZ for 24 hresults in a smaller induction of mda-6 expression and reduced growthinhibition (Fisher et al., 1985; Jiang & Fisher, 1993; Jiang et al.,1993). Enhanced mda-6 expression and terminal cell differentiation arealso produced in additional human melanoma cells treated with IFN-β+MEZ,including FO-1, SH-1, LO-1, WM-239 and WM-278 (FIG. 32). mda-6 isexpressed at higher de novo levels in a low-density SV40-transformedhuman melanocyte culture (FM516-SV) (Melber et al., 1989) than inlow-density and the majority of high-density melanoma cultures (FIG. 32and data not shown). mda-6 levels are increased and melanoma growth isinhibited after 24 h treatment with IFN-β+MEZ and 96 h treatment withthe inducers results in terminal differentiation in the human melanomacultures (FIG. 32 and data not shown). In contrast, although mda-6expression is enhanced by 24 h in IFN-β+MEZ-treated FM516-SV cells,growth is only marginally reduced (≦15%) (FIG. 32 and data not shown).Unlike human melanoma cells, 96 h exposure of FM516-SV cells toIFN-β+MEZ does not result in terminal differentiation in the majority oftreated cells (data not shown).

Elevated mda-6 expression persists in HO-1 cells, and other melanomacells, induced to terminally differentiate by continuous exposure toIFN-β+MEZ for 96 h, whereas HO-1 cells, and additional melanomacultures, treated singly with IFN-β or MEZ for 96 h recover from growthsuppression and contain similar levels of mda-6 as control cells (FIGS.31A-E and data not shown). Previous studies indicate that growth of HO-1cells for 24 h in IFN-β+MEZ or MEZ, but not IFN-β, followed by removalof the inducing agent(s) and growth for an additional 72 h in completemedium results in sustained growth inhibition (Jiang et al., 1993).Under these experimental conditions, mda-6 expression remains elevatedwith both types of treatment with the highest expression occurring withIFN-β+MEZ that also induces the greatest residual growth inhibition(Jiang et al., 1993) (FIGS. 31A-E).

Even without treatment with differentiation-inducing agents, HO-1 cellsgrown to high density (FIGS. 31A-E) or grown in the absence of serum(FIGS. 31A-E) express elevated levels of mda-6 mRNA. The induction ofenhanced mda-6 mRNA levels is rapid, occurring within 15 min treatment(FIGS. 31A-E). Elevated mda-6 expression is also evident in IFN-β+MEZtreated HO-1 cells simultaneously culured in the presence ofcycloheximide (data not shown) and following a 2 h exposure to 25 μg/mlof the alkylating carcinogen, methyl methanesulfonate (data not shown).These observations suggest a direct relationship between mda-6expression and growth suppression in human melanoma cells.

Inverse Relationship Between p21 and Wild-type p53 Levels During GrowthArrest and Differentiation in HO-1 Human Melanoma Cells

Treatment of HO-1 cells with IFN-β+MEZ results in rapid growth arrest,that is apparent within 24 h (Fisher et al., 1985; Jiang et al., 1993).In contrast, IFN-β and MEZ alone result in smaller changes in growth inHO-1 cells (Fisher et al., 1985; Jiang et al., 1993). Analysis of p53mRNA levels in IFN-β, MEZ and IFN-β+MEZ treated HO-1 cells indicate nosignificant change after 24 h, but significant inhibition is observed inp53 expression that is maximum in IFN-β+MEZ (96 h) treated terminallydifferentiated HO-1 cells (Jiang et al., in preparation). MEZ alsoinduces a reduction in p53 mRNA levels in 96 h treated HO-1 cells,whereas no change in p53 mRNA occurs in HO-1 cells treated with IFN-βfor 96 h (Jiang et al., in preparation). Since the kinetics ofsuppression in p53 gene expression is the opposite of that observed withmda-6 expression in differentiation inducer treated HO-1 cells,experiments were conducted to determine the effects of growthsuppression and terminal differentiation on p53 and p21 protein levels.Immunoprecipitation analysis of p53 under conditions preventing proteindenaturization (<1% SDS) with monoclonal antibodies Ab1 (PAb421;Oncogene Sciences), that identifies both wild-type and mutant p53, andAb3 (PAb240; Oncogene Sciences) that recognizes mutant p53, indicatethat HO-1 cells contain a wild-type p53 protein (data not shown). Awild-type p53 protein is also present in a number of other cell typesevaluated in the present study, including FM516-SV, LO-1, SH-1 and FO-1,whereas WM239 cells contain a mutant p53 (data not shown). To rule outpotential artifacts, immunoprecipitation studies with Ab1 and Ab3 wereperformed with labeled extracts from cell lines with known p53 status,including MeWo (previously shown to contain a mutant p53 by sequenceanalysis) (Loganzo et al., 1994), Saos-2 (p53-null phenotype), humanskin fibroblasts (wild-type p53) and SW480 colon carcinoma cells (mutantp53) (data not shown). The current results are in agreement with severalrecent studies (Volkenandt et al., 1991; Castresana et al., 1993;Greenblatt et al., 1994; Montano et al., 1994; Loganzo et al., 1994)indicating that p53 mutations are rare in human melanoma and themajority of human melanomas contain a wild-type as opposed to a mutantp53 protein.

To determine the effect of the various inducing agents on p53 and p21protein levels the following experiment was performed. HO-1 cells weregrown in inducer-free medium (control), IFN-β (2000 units/ml), MEZ (10ng/ml) or IFN-β+MEZ (2000 units/ml+10 ng/ml) for 24, 48, 72 or 96 h,cells were labeled with ³⁵ S-methionine and cell lysates were preparedand analyzed by immunoprecipitation analyses using Ab1 (PAb421), p21(WAF1/CIP1, Santa Cruz Biotechnology; and rabbit polyclonal antibodiesprepared against mda-6 peptides) and actin (Oncogene Sciences Inc.)(FIGS. 33 and 34). As observed with mRNA levels, no significant changein wild-type p53 protein occurs in HO-1 cells treated for 24 h withIFN-β, MEZ or IFN-β+MEZ (FIG. 33). In contrast, p21 mRNA and protein areinduced in HO-1 cells, with IFN-β+MEZ>MEZ>IFN-β (FIGS. 31A-E and 33). Asseen with mRNA levels, the concentration of wild-type p53 proteindecreases and p21 protein increases over a 96 h period in MEZ andIFN-β+MEZ treated HO-1 cells (FIG. 34). Increases in p21 protein arealso seen in HO-1 cells treated with IFN-β for 48, 72 or 96 h, whereasno change in wild-type p53 protein occurs over the same period insimilarly treated cells (FIG. 34). These results indicate that inductionof p21 can occur without increases in wild-type p53 protein (IFN-βtreated cells) and elevated levels of p21 under conditions of residualgrowth arrest and/or terminal differentiation correlate with a reductionin wild-type p53 protein in HO-1 melanoma cells.

Inverse Relationship Between mda-6 Expression and Evolution from NormalMelanocyte to Metastatic Melanoma

A prediction of applicants' melanoma aberrant differentiation model isthat normal melanocytes should express elevated levels of specific mdagenes with progressively less expression in primary and metastaticmelanoma cells. As shown in FIG. 32, the level of mda-6 is higher in anactively growing low-density SV40-transformed human melanocyte culture(FM516-SV) than in corresponding high-density proliferating humanmelanoma cells. The level of mda-6 is variably increased in all of themelanomas treated with IFN-β+MEZ (FIG. 32). Induction of mda-6 occurs inlogarithmically growing FM516-SV cells treated with IFN-β+MEZ. However,only the melanoma cells become terminally differentiated followinggrowth for 96 h in IFN-β+MEZ (data not shown). To evaluate mda-6 levelsas a function of melanoma evolution, mda-6 and GAPDH (internal RNAexpression standard) levels were determined by comparative RT-PCR inactively growing melanocytes (5 samples), a dysplastic nevus (1 sample),an SV40-transformed immortalized melanocyte culture (1 sample), RGP (1sample) and early VGP (4 samples) primary melanomas and metastaticmelanomas (6 samples) (FIG. 35). The highest levels of mda-6 are foundin actively growing melanocytes and the dysplastic nevus and the lowestrelative levels of mda-6 are present in primary and metastaticmelanomas. The difference in relative mda-6 expression (as a function ofGAPDH expression) determined by comparative RT-PCR indicates thatactively growing normal melanocytes express on average >4-fold moremda-6 than actively growing metastatic melanoma cells (P <0.01). Theseresults suggest an inverse correlation between levels of mda-6expression and human melanoma evolution.

Reduced Expression of mda-6 in Matrigel-progressed Primary HumanMelanoma Cultures

The ability to study human melanoma progression by comparison ofsequentially obtained cell lines established from the same patient isvery limited since removal of the vast majority of RGP or thin VGPprimary melanomas results in cure (Herlyn, 1990; Kerbel, 1990; Clark,1991). Consequently, the derivation of genetically related variants fromsuch tumors that express a biologically more aggressive phenotype mustbe obtained under experimental selections. One such method is"Matrigel-assisted" tumorigenic growth (Kobayashi et al., 1994). Forexample, the early stage primary melanoma cell lines known as WM35,WM1341B and WM793 are non or poorly tumorigenic in nude mice incomparison to the great majority of advanced stage human melanomas(Kobayashi et al., 1994). However, co-injection of the early stage celllines with Matrigel, a reconstituted basement membrane extract permitsrapid tumor growth in nude mice and the derivation of sublines that willreadily grow as solid tumors in secondary nude mouse recipients, even inthe absence of Matrigel co-injection (Kobayashi et al., 1994). Thesetumorigenic Matrigel progressed sublines can be compared to thenon/poorly tumorigenic parental cell lines for various properties suchas relative mda-6 expression. As shown in FIG. 36, the level of mda-6 asdetermined by RT-PCR in Matrigel progressed sublines of WM35, WM1341Band WM793 are variably reduced in comparison with the originalpatient-derived cell lines. The relative degree of suppression in mda-6is greatest in the WM793 series, that also display a more progressedphenotype as indicated by a low-level of de novo tumorigenic potentialin nude mice in the absence of Matrigel (Kobayashi et al., 1994). Thesmallest reduction in expression occurs in the Matrigel-progressed WM35RGP primary melanoma cells (FIG. 36). Treatment of parental andMatrigel-progressed RGP and early VGP melanomas with IFN-β+MEZ resultsin increased mda-6 expression (determined by Northern blotting) andgrowth suppression (data not shown). These observations provide furthersupport for an inverse relationship between mda-6 expression and humanmelanoma progression and melanoma growth.

Increased Expression of mda-6 in C8161 Cells Containing a Normal HumanChromosome 6

Insertion of a normal human chromosome 6 into tumorigenic and metastaticC8161 human melanoma cells results in an extinction of the metastaticphenotype with a retention of tumorigenic potential (Table 5) (Welch etal., 1994). These results suggest that chromosome 6 contains asuppressor gene that can revert C8161 cells to a less-progressed stagein melanoma evolution (Welch et al., 1994). If mda-6 expressioncorrelates directly with states of melanoma progression, it would bepredicted that actively growing C8161 cells should produce lower levelsof mda-6 than actively growing chromosome 6 containing C8161 cells. Asanticipated, expression of mda-6 increases in three independentlyderived chromosome 6 containing C8161 cells (FIG. 37). In addition,treatment of C8161 and neo6/C8161 hybrid clones with IFN-β+MEZ for 96 hrresults in growth suppression (Table 5) and increased mda-6 mRNAexpression (FIG. 37). These observations indicate a direct relationshipbetween growth suppression and metastatic suppression in human melanomacells and elevated expression of mda-6 (p21).

                  TABLE 5                                                         ______________________________________                                        Properties of human melanoma cells containing a                                 microcell-transferred chromosome 6                                          Cell Types.sup.1 Tumorigenicity.sup.2                                                                      Metastasis.sup.3                                 ______________________________________                                          C8161 + +                                                                     C8161/6.1 (neo6/C8161.1) + -                                                  C8161/6.2 (neo6/C8161.2) + -                                                  C8161/6.3 (neo6/C8161.3) + -                                                ______________________________________                                                                          %                                                growth                                                                     Cell Types.sup.1 nm23.sup.4 mda-6.sup.5 inhibition.sup.6                    ______________________________________                                          C8161 +/- +/- 92                                                              C8161/6.1 (neo6/C8161.1) +++ +++ 67                                           C8161/6.2 (neo6/C8161.2) + to ++ +++ 86                                       C8161/6.3 (neo6/C8161.3) ++ ++ 89                                           ______________________________________                                         .sup.1 A neomycintagged normal human chromosome 6 was transferred into        C8161 by microcellmediated chromosome transfer as previously described        (Welch et al., 1994). Retention of chromosome 6 in cell lines and tumor       tissue was verified using PCRRFLP with D6S87 and D6S37.                       .sup.2 Tumorigenicity was determined by injection of 1 × 10.sup.6 t     1 × 10.sup.7 cells subcutaneously or intradermally into the             dorsolateral flank of 31/2 to 4week old, female athymic nude mice (Harlan     Sprague Dawley). +, palpable tumors form within 2 weeks after injection.      .sup.3 Development of lung metastases following s.c., i.d. (1 ×         10.sup.6 cells) or i.v. (1 × 10.sup.6 cells) injection of tumor         cells. Spontaneous metastases were evaluated in the same mice in which        tumorigenicity was assessed. At necropsy all organs were examined for         grossly visible nodules. Lack of metastases was verified by histologic        analysis of randomly submitted samples. Experimental metastases was           measured in mice receiving a single cell suspension of viable  # cells        into the lateral tail vein. Table 1 Legend (Continued):                       .sup.4 Expression of nm23H1 determined using Northern blot of total RNA       (10-20 μg) using the 0.9 kb fragment of nm23H1 (Welch et al., 1994).       .sup.5 Expression of mda6/WAF1/CIP1 determined using Northern blot of         total RNA (15 μg) using the mda6 cDNA insert (Jiang & Fisher, 1993).       .sup.6 Cell growth was determined by counting cells following 96 hr           continuous treatment with IFNβ (1000 units/ml) + MEZ (10 ng/ml).         Results are the average percent growth inhibition verus untreated control     cultures for triplicate plates that varied by ≦10% (Jiang et al.,      1993).                                                                   

Experimental Discussion

The specific genomic changes that mediate melanoma development andprogression remain to be elucidated (Herlyn, 1990; Kerbel, 1990; Clark,1991). To directly approach this question and to begin to identify andclone genes involved in growth control and differentiation in humanmelanoma cells applicants have used subtraction hybridization (Jiang &Fisher, 1993; Jiang et al., 1994a). cDNA libraries were constructed fromuntreated HO-1 human melanoma cells and these cDNAs were subtracted fromcDNA libraries prepared from HO-1 cells treated with the combination ofIFN-β plus MEZ that induces an irreversible loss of proliferativeability and terminal differentiation (Fisher et al., 1985; Jiang &Fisher, 1993; Jiang et al., 1993, 1994a). This approach has resulted inthe identification of several novel mda cDNA clones that displayenhanced expression as a function of growth suppression and induction ofterminal differentiation in human melanoma cells (Jiang & Fisher, 1993;Jiang et al., 1994a). In the present study applicants have analyzedmda-6 (Jiang & Fisher, 1993; Jiang et al., 1994a), whose open readingframe sequence (FIG. 30) is identical to the genes WAF1, CIP1 and SDI1(El-Deiry et al., 1993; Harper et al., 1993; Noda et al., 1994). WAF1was cloned using a strategy designed to identify inducible down-streamgenes that are directly controlled by and might mediate the growthsuppressing activity of the tumor suppressor gene p53 (El-Deiry et al.,1993). Introduction of WAF1 cDNA into human brain, lung and colon tumorcell cultures results in growth suppression (El-Deiry et al., 1993). Inaddition, WAF1 is induced by DNA damage in wild-type p53-containingcells and during the process of p53-associated G₁ arrest or apoptosis(El-Deiry et al., 1994). CIP1 was identified using an improvedtwo-hybrid system and encodes a 21-kDa product that is a potentinhibitor of cyclin-dependent kinases (Harper et al., 1993). CIP1induces growth suppression in normal diploid fibroblasts but onlymarginally inhibits growth in SV40-transformed diploid fibroblasts(Harper et al., 1993). SDI1 was identified and cloned from senescenthuman fibroblasts using an expression screening strategy designed todetect cDNAs that could prevent young fibroblasts from initiating DNAsynthesis (Noda et al., 1994). The current studies indicate that mda-6(WAF1/CIP1/SDI1) expression is also related to growth regulation inhuman melanoma cells and its reduced expression may contribute to theprogressive changes observed in the evolution of melanocytes intometastatic melanomas.

Cancer is a progressive disease often affected by the altered expressionof oncogenes that promote the cancer phenotype and tumor suppressorgenes that inhibit the cancer phenotype (for review see: Fisher, 1984;Bishop, 1991; Vogelstein & Kinzler, 1992; Lane, 1992). Recent evidenceindicates that the tumor suppressor gene p53 is a major component in thecarcinogenic process (for review see: Vogelstein & Kinzler, 1992; Lane,1992; Greenblatt et al., 1994). Inactivation of wild type p53 orexpression of a mutant p53 phenotype has been found in a large number ofhuman cancer subtypes (for review see: Vogelstein & Kinzler, 1992; Lane,1992; Greenblatt et al., 1994). Intensive effort has been directedtoward elucidating the mechanism by which wild type p53 regulates cellgrowth and prevents expression of the tumorigenic phenotype and theprocess by which p53 inactivation or mutagenic changes promote theseprocesses. In this context, the identification of genes positivelycontrolled by wild type p53, such as WAF1/CIP1, may prove important indefining the mechanism of action of this critical tumor suppressor geneand provide information about the process of tumor progression.

Metastatic human melanomas appear to be unique among human cancers intheir low frequency of p53 mutations and the prevalence of wild-type p53protein in advanced cancers (Volkenandt et al., 1991; Castresana et al.,1993; Greenblatt et al., 1994; Montano et al., 1994; Lu & Kerbel, 1994).Studies by Loganzo et al. (1994) show that metastatic melanoma cellscontain two- to 20-fold more p53 protein, in the majority of samplesrepresenting wild-type p53, than do melanocytes. Similarly, a largeproportion of the human melanoma cell lines presently analyzed alsocontain wild-type p53 protein. In normal melanocytes, the level of mda-6(p21) is higher than in metastatic melanomas, even though metastaticmelanoma may contain more p53 (FIG. 35) (Loganzo et al., 1994). Theincreased level of p53 protein in melanoma cells appears to be aconsequence of stabilization of the protein, i.e., the half-life is two-to five-fold greater than in melanocytes, irrespective of whether theycontain wild-type or mutant-p53 (Loganzo et al., 1994). Thestabilization of wt p53 protein in human melanoma cells does not resultfrom the binding of this protein to either MDM2 or heat shock protein(Loganzo et al., 1994). The mechanism underlying this stabilization ofwild-type p53 in metastatic melanomas is not presently known, but itmight reflect a defective regulation of p53 that could allow these tumorcells to escape cell cycle arrest even in the presence of elevated p53.In fact, disturbances in p53 expression are a common occurrence in humanmelanomas and these abnormalities increase with progression (for reviewsee: Lu & Kerbel, 1994). These findings suggest that melanoma mayrepresent a novel malignancy, in that it can coexist and evolve to moreaggressive stages even in the presence of elevated levels of nuclearlocalized wt p53 protein. However, it is also possible that thewild-type p53 protein in metastatic human melanoma cells is functionallyinactive (perhaps by interacting with other melanoma proteins) or thewild-type p53 protein is normal, i.e., can both bind andtranscriptionally activate target genes, but the downstream genesnormally responsive to wild-type p53 are defective in metastatic humanmelanoma. The inability of wild-type p53 to elevate mda-6 levels inmetastatic melanoma, and consequently to induce proliferative control,could directly contribute to the increased instability of the evolvingand progressing melanoma (Livingstone et al., 1992; Yin et al., 1992; Lu& Kerbel, 1994).

In human melanoma, mda-6 is induced rapidly (within 15 min) and remainselevated following serum starvation as well as remaining elevated duringterminal differentiation (FIGS. 31A-E). In contrast, glioblastomamultiforme cells blocked in G₀ by serum starvation or blocked in G₁ bymimosine treatment do not display increased levels of wild-type p53 orWAF1/CIP1 (El-Deiry et al., 1994). Induction of apoptosis after IL3withdrawal, which also does not increase wild-type p53 levels, and DNAdamage of cells containing a mutant p53 does not result in elevatedlevels of WAF1/CIP1 (El-Deiry et al., 1994). Recent studies suggest thatwild-type p53 may not be obligatory for induction of WAF1/CIP1 (p21).Michieli et al. (1994) document a transient induction of WAF1/CIP1following stimulation of growth arrested cells either containing orlacking wild-type p53 (fibroblasts from p53 knock out mice lacking p53protein) with various growth factors. Jiang et al. (1994b) demonstratethat treatment of human promyelocytic leukemia HL-60 cells, which do notexpress p53, with agents inducing either granulocytic ormacrophage/monocyte differentiation results in the rapid activation andpersistent expression of mda-6 (p21). Steinman et al. (1994) alsoprovide evidence that p21 is upregulated during induction ofdifferentiation in a number of cell types, including hematopoietic andhepatoma cells, in a p53-independent pathway. All three of these studiesalso provide evidence that p21 is an immediate early response gene thatis induced in the absence of protein synthesis.

The present study provides additional evidence indicating that inductionof p21 expression is independent of wild-type p53 expression in humanmelanoma cells. An interesting, yet somewhat paradoxical observation, isthe temporal decrease in wild-type p53 protein with a correspondingincrease in p21 protein during the process of growth arrest andinduction of terminal differentiation in HO-1 melanoma cells (FIG. 34).In a number of cell culture model systems, p53 mRNA decreases as afunction of growth suppression and the induction of differentiation(Shen et al., 1983; Mercer et al., 1984; Dony et al., 1985; Shobat etal., 1987; Khochbin et al., 1988; Richon et al., 1989; Hayes et al.,1991). Wild type p53 displays sequence-specific DNA-binding activity,sequence-specific transcriptional activation and induces growthsuppression in a number of cell types, whereas all of these propertiesare lost in various mutant forms of the p53 protein (Ron, 1994;Pietenpol et al., 1994). The reduced levels of mda-6 (p21) in activelygrowing melanoma, even in the presence of high levels of wild type p53,and the elevations in p21 levels following wild type p53 suppressionsuggest that high levels of p21 expression may not be compatible withhigh levels of wild type p53 in human melanoma. This may occur becausewild type p53 is inducing a downstream gene that may directly orindirectly modify p21 expression. The induction of growth arrest andterminal differentiation program by IFN-β+MEZ in HO-1 cells may resultin genotypic changes that mediate an inhibition of wild type p53expression and consequently the absence of the downstream inhibitor ofp21 expression. Alternatively, the wild type p53 protein that is presentin metastatic melanoma may be functionally inactive or a downstreampathway modified by wild type p53 may be aberrant in progressingmelanoma cells. In this context, the inverse relationship observedbetween wild type p53 and p21 protein levels may be associated with butnot functionally relevant to growth arrest and terminal differentiationinduced by MEZ and IFN-β+MEZ.

In the present study applicants have not directly assayed for the effectof mda-6 on progression in human melanoma. However, experimentsutilizing chromosome 6 containing C8161. cells provide indirect evidencethat mda-6 (WAF1/CIP1/SDI1), which is located on chromosome 6p21.2(El-Deiry et al., 1993), can directly modulate in vivo tumor growth andmetastatic progression in human melanoma cells. Transfer of a normalchromosome 6 into the human melanoma cell lines UACC-903 and UACC-091results in a suppression of tumorigenicity (Trent et al., 1990; Milikinet al., 1991), whereas microcell transfer of a normal chromosome 6 intothe tumorigenic and metastatic human melanoma cell line C8161 results ina suppression of metastasis but a retention of tumorigenic potential(Welch et al., 1994). C8161 microcell hybrids (neo6/C8161) also displaya small but significant increase in tumor latency time and have slowertumor growth rates in vivo than parental C8161 cells (Welch et al.,1994). However, even 30 additional weeks in animals does not result inmetastatic lesions in mice injected with any of the three independentneo6/C8161 hybrids (Welch et al., 1994). In the present study,applicants demonstrate that C8161 cells contain lower levels of mda-6than three neo6/C8161 hybrids. Treatment with the combination ofIFN-β+MEZ results in enhanced expression of mda-6 and growth suppressionin parental C8161 and all three neo6/C8161 hybrid clones. These resultsstrongly implicate mda-6 as a potential mediator of growth control andmetastatic progression in human melanoma cells. mda-6 has now beencloned into the pMAMneo vector, that allows inducible expression of theinserted gene by dexamethasone (DEX) and which also contains a neomycinresistance gene permitting clonal isolation in G418 in the absence ofmda-6 expression (Jiang et al., in preparation). Electroporation of thisgene into C8161 cells has resulted in the isolation of G418-resistantcultures containing the pMAMneo-mda-6 construct. When grown in thepresence of DEX, mda-6 expression is induced and growth is inhibited,whereas no growth suppression or mda-6 expression occurs in the absenceof DEX (Jiang et al., in preparation). These genetically modified C8161cells, and melanoma cells containing an inducible antisense mda-6 gene,will prove useful in directly determining the effect of mda-6 expressionon melanoma growth, differentiation and tumor progression.

Recent studies indicate that p21, the protein encoded by mda-6(WAF1/CIP1/SDI1), is a major contributor to many important cellularprocesses, including cell cycle regulation, cell growth, DNA repair andDNA replication (Xiong et al., 1992, 1993a, 1993b; Gu et al., 1993;Harper et al., 1993; El-Deiry et al., 1993, 1994; Waga et al., 1994; Liet al., 1994). p21 is a ubiquitous inhibitor of all cyclin-dependentkinases (Xiong et al., 1993b). The levels of p21 vary depending on thespecific stage of the cell cycle and the interaction between p21 andspecific cyclin-CDK enzymes appears to occur when these enzymes functionin cell cycle control (Li et al., 1994). In the present study applicantsdemonstrate that mda-6 (WAF1/CIP1/SDI1) expression inversely correlateswith growth, differentiation and progression in human melanoma cells.These observations suggest that p21, that is encoded by mda-6, canaffect cellular differentiation and neoplastic progression in humanmelanoma cells. The relative levels of mda-6 are higher in growing humanmelanocyte and nevus cell lines than in RGP, VGP and metastaticmelanomas, suggesting the possibility that expression of this suppressorprotein may negatively regulate tumor progression. This possibility issupported by the observation that mda-6 expression decreases inMatrigel-progressed early VGP melanomas and mda-6 expression increasesin chromosome 6 metastasis-suppressed C8161 melanoma cells. A directrelationship between mda-6 expression and melanoma growth anddifferentiation is indicated by the ability of IFN-β+MEZ to inducegrowth suppression and with continuous exposure terminal differentiationin human melanoma cells. Apparently, increased levels of mda-6 can betolerated by human melanoma cells resulting in or correlating withgrowth arrest. However, the persistence of elevated levels of p21 interminally differentiated human melanoma cells may be necessary toprevent cells from reentering the cell cycle, a mandatory requirementfor terminal cell differentiation. In this context, identification ofagent(s) that can increase mda-6 (WAF1/CIP1/SDI1) expression inmetastatic human melanoma cells may prove beneficial in the therapy ofthis malignancy by directly inducing an irreversible loss ofproliferative capacity and terminal cell differentiation.

Materials and Methods

Cell Lines and Culture Conditions

HO-1 melanoma cells were established from a melanotic melanoma obtainedfrom a 49-year-old female and were used between passage 125 and 160(Fisher et al., 1985,1986; Giovanella et al., 1976). FM516-SV is anormal human melanocyte culture immortalized by the SV40 T-antigen gene(Melber et al., 1989). Normal human melanocytes, FM713, FM723, FM741,FM841 and FM793, and a dysplastic nevus, N3153, were established frompatients as described previously (Mancianti et al., 1988). WM35 wasderived from an RGP primary human melanoma and WM278, WM1341B, WM793 andWM902B were derived from early VGP primary human melanomas (Herlyn,1990; Herlyn et al., 1989). WM Matrigel progressed WM35, WM1341B andWM793 cells, referred to as P1-N1 and P2-N1 that indicates first andsecond passage through nude mice injected with the appropriate cell typeplus Matrigel, were developed as described (MacDougall et al., 1993;Kobayashi et al., 1994). C8161 is a highly metastatic amelanotic humanmelanoma cell line derived from an abdominal wall metastasis (Welch etal., 1991). C8161 clones containing a normal human chromosome 6,designated C8161/6.1 (neo6/C8161.1), C8161/6.2 (neo6/C8161.2) andC8161/6.3 (neo6/C8161.3), were established as described (Welch et al.,1994). Additional human melanoma cell lines isolated from patients withmetastatic melanomas included FO-1, LO-1, SH-1, WM239 and WM239A(Giovanella et al., 1976; Fisher et al., 1985; Herlyn et al., 1989;Herlyn, 1990). Media and culture conditions used to grow the variouscell types are described in the indicated references.

Cell Growth and Terminal Cell Differentiation Assays

Cell growth and terminal differentiation assays were performed asdescribed previously (Fisher et al., 1985, 1986; Jiang et al., 1993).Induction of terminal cell differentiation following 96 h growth inIFN-β+MEZ was monitored by an irreversible loss of proliferativepotential without a loss of cell viability (Fisher et al., 1985, 1986;Jiang et al., 1993). Briefly, cells were grown for 96 h in the presenceof the various inducing agents, the inducers were removed and cultureswere washed 3× in medium without serum followed by the addition ofinducer-free medium. Cultures were incubated for an additional 3, 6 and10 d with a medium change without inducers every 3 d. Cell numbers weredetermined at d 4, 7, 10 and 14 after the beginning of the assay.Terminal differentiation was indicated by the absence of cell numberincreases and the retention of cell viability over the 7 to 14 dincubation in the absence of inducers.

Subtraction Hybridization, RACE and Sequence Analysis

Identification and cloning of mda-6 by subtraction hybridization wasachieved as described (Jiang & Fisher, 1993). A full-length mda-6 cDNAwas isolated by screening a differentiation inducer-treated HO-1 cDNAlibrary (Jiang & Fisher, 1993) and using the procedure of rapidamplification of cDNA ends (RACE) as described (Frohman et al., 1988;Loh et al., 1989; Ohara et al., 1989). Sequence analysis was determinedas described (Sanger et al., 1977; Su et al., 1993). The completesequence of mda-6 consists of 2149 nucleotides (U09579 in GenBank) andthe longest open reading frame starting with a methionine codon atposition 95 in the nucleotide sequence encodes a 164-amino acidpolypeptide. Compared with the current protein database releases usingthe GCG/TFASTA program, the predicted mda-6 amino acid sequence isidentical to the sequences of WAF1/CIP1/SDI1 (El-Deiry et al., 1993;Harper et al., 1993; Noda et al., 1994).

RNA Isolation, Northern Blotting and RT-PCR

Total cytoplasmic RNA was isolated and Northern blotting hybridizationwas performed as described (Reddy et al., 1991; Su et al., 1991; Jianget al., 1992). Ten μg of RNA was denatured with glyoxal/DMSO,electrophoresed on 1.0% agarose gels, transferred to nylon membranes andhybridized to a ³² P-labeled p53 probe (Baker et al., 1990). The nylonmembrane was stripped and hybridized to a ³² P-labeled mda-6 probe(Jiang and Fisher, 1993) and then after a second stripping the membranewas hybridized to a ³² P-labeled rat GAPDH probe (Fort et al., 1985), asdescribed previously (Reddy et al., 1991; Su et al., 1991; Jiang et al.,1992). Following hybridization, the filters were washed and exposed forautoradiography (Reddy et al., 1991; Su et al., 1991; Jiang et al.,1992). mda-6 and GAPDH gene expression were also determined by reversetranscription-polymerase chain reaction (RT-PCR) as described (Adollahiet al., 1991; Lin et al., 1994; Jiang et al., 1994b). Total cytoplasmicRNA was treated with 0.5 units DNase (Boehringer-MannheimBiochemicals)/μg RNA in 15% glycerol, 10 mM Tris, pH 7.5, 2.5 mM MgCl₂,0.1 mM EDTA, 80 mM KCl, 1 mM CaCl₂ and 1 unit/ml RNasin (Promega) at 30°C. for 10 min. RNA was extracted with phenol-chloroform, precipitatedwith sodium acetate/ethanol and RNA pellets were resuspended indiethylpyrocarbonate-treated H₂ O. One μg of total RNA was reversetranscribed with 200 units of murine leukemia virus reversetranscriptase (Bethesda Research Laboratories) in 20 μl containing 1 mmdeoxyribonucleotide triphosphates, 4 mM MgCl2, 10 mM Tris, pH 8.3, 50 mMKCl, 0.001% gelatin and 0.2 μg oligo-dT primer. Samples were adjusted to100 μl with buffer containing 0.2 mM deoxyribonucleotide triphosphates,2 mM MgCl2, 10 mM Tris, pH 8.3, 50 mM KCl and 0.001% gelatin. Fifty pmolof each primer, 1.5 units Taq DNA polymerase (Perkin-Elmer Cetus) wereadded and samples were covered with mineral oil, heated at 95° C. for 5min and subjected to 25 cycles of PCR in a Perkin-Elmer Thermal Cyclerusing 1 min denaturation at 94° C., 2 min annealing at 55° C. and 3 minpolymerization at 72° C. After extraction with chloroform, 20 μl ofproducts were electrophoresed, blotted onto nylon filters and hybridizedwith an mda-6 or GAPDH specific probe. The mda-6 primers were 5' to 3'CTCCAAGTACACTAAGCACT (SEQ ID NO:28) and TAGTTCTACCTCAGGCAGCT (SEQ IDNO:29) (corresponding to nt 1527 to 1546) (GenBank accession numberU09579); and the GAPDH primers were 5' to 3' TCTTACTCCTTGGAGGCCATG (SEQID NO:30) and CGTCTTCACCACCACCATGGAGAA (SEQ ID NO:31) (corresponding tont 1070 to 1053) (Tokunaga et al., 1987).

Immunoprecipitation Analyses

Immunoprecipitation analyses were performed as described previously(Duigou et al., 1991; Su et al., 1993; Jiang et al., 1994b).Logarithmically growing HO-1 cells were either untreated or treated for24, 48, 72 or 96 h with IFN-β (2000 units/ml), MEZ (10 ng/ml) orIFN-β+MEZ (2000 units/ml+10 ng/ml) in 10-cm plates. Cultures werestarved of methionine for 1 h at 37° C. in methionine-free medium, cellswere concentrated by pelleting and labeled for 1 h (p53) or 4 h (p21 andActin) at 37° in 1 ml of the same medium with 100 μCi of [³⁵ S] (NEN;Express ³⁵ S). After labeling, the cells were washed twice with ice-coldphosphate-buffered saline and lysed for 1 h on ice by the addition ofRIPC (20 mM Tris-base, pH 7.5, 500 mM NaCl, 0.05% Nonidet P-40, 100μg/ml phenylmethylsulfonyl fluoride and 0.02% sodium azide). The lysatewas clarified by centrifugation in an Eppendorf microfuge at 10,000×gfor 10 min at 4° C. HO-1 samples containing 4×10⁶ counts were incubatedwith 2 μg of p53 monoclonal antibody (Ab1; PAb421) (Oncogene Sciences),WAF1/CIP1(C-19) (Santa Cruz Biotechnology) (or MDA-6 peptide-derived)rabbit polyclonal IgG or actin monoclonal antibody (oncogene Sciences)with rocking at 4° C. for 24 h. Labeled cell lysates were also preparedfrom FO-1, LO-1, SH-1, WM239, FM516-SV, SW480, MeWo, human skinfibroblasts and Saos-2 cells. Samples containing 4×10⁶ counts wereincubated with 2 μg of the p53 monoclonal antibody Ab1 (PAb421) or Ab3(PAb240) (Oncogene Sciences). The next day, 30 μl (packed volume) ofprotein G-agarose (Oncogene Sciences) was added to each tube, andincubation with rocking at 4° C. continued for another hour. The proteinG pellets were then washed five times with 1 ml of ice-coldRIPC:phosphate-buffered saline (1:1, v/v). Thirty μl of sodium dodecylsulfate-polyacrylamide gel electrophoresis buffer were added to thepellets, and the sample was heated at 87° C. for 3 min. The samples wereloaded onto an 10% polyacrylamide gel and run overnight at 40 V. The gelcontained Rainbow protein markers (Amersham) for sizing. Gels were fixedwith 10% acetic acid plus 10% methanol for 30 min, incubated in DMSO for30 min, incubated with 10% 2,5-diphenyloxazole in DMSO for 30 min,washed three times with cold water (10 min each), dried, and exposed tofilm.

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Volkenandt, M., Schlegel, U., Nanus, D. M. & Albino, A. P. (1991).Pigment Cell Res., 4, 35-40.

Waga, S., Hannon, G. J., Beach, D. & Stillman, B. (1994). Nature, 369,574-578.

Waxman, S., Rossi, G. B. & Takaku, F. (1988). The Status ofDifferentiation Therapy of Cancer, vol. I, Raven Press Inc.: New York.

Waxman, S., Rossi, G. B. & Takaku, F. (1991). The Status ofDifferentiation Therapy of Cancer, vol. II, Raven Press Inc.: New York.

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Eighth Series of Experiments

Properties of mda-7

mda-7 is a novel cDNA (it has no sequence homology with previouslyreported genes in the various DNA data bases). The full-length cDNAcontains 1718 nt, the open reading frame extends from nt 275 to no 895and encodes a protein of 206 amino acids, containing a membrane domainand three potential glycosylation sites.

Expression in HO-1 Human Melanoma Cells

Increased expression of mda-7 after 24 hr treatment of HO-1 cells withrecombinant human fibroblast interferon (IFN-β) (2000 units/ml), MEZ (10ng/ml) and to the greatest extent with IFN-β+MEZ (2000 units/ml+10ng/ml).

Increased expression of mda-7 is observed in HO-1 cells treated for 96hr with IFN-β (2000 units/ml), MEZ (10 ng/ml), MPA (3 μM), IFN-β+IFN-γ(1000 units/ml+1000 units/ml), IFN-β+MEZ (2000 units/ml+10 ng/ml,MPA+MEZ (3 μM+10 ng/ml) and RA+MEZ (2.5 μM+10 ng/ml). Maximum inductionis observed with IFN-β+MEZ followed by MPA+MEZ and IFN-β+IFN-γ.

The relative level of mda-7 induction correlates with the degree ofgrowth suppression observed HO-1 cells treated with the various growthand differentiation modulating agents. The greatest increase inexpression is observed in cells induced to irreversibly loseproliferative capacity and become terminally differentiated by treatmentwith IFN-β+MEZ.

Expression in Additional Human Melanoma Cells

Increased expression of mda-7 occurs in HO-1, C8161, C8161/6.3 (a C8161human melanoma cell clone containing an inserted normal human chromosome6: These cells are tumorigenic in nude mice, but unlike parental C8161cells they are non-metastatic), FO-1, LO-1, SH-1, WM278 and WM239 humanmelanoma cells treated with IFN-β+MEZ for 24 hr. This gene isconstitutively expressed in immortalized human melanocytes FM5169(transformed by SV40). However, no increase in expression is observed inFM5169 following IFN-β+MEZ treatment for 24 hr.

mda-7 is either variably expressed or variably induced in all humanmelanoma cells treated with IFN-β+MEZ. In contrast, although this geneis expressed in melanocytes, no change in expression is observedfollowing a 24 hr treatment with IFN-β+MEZ.

Expression in Human Neuroblastoma Cells

mda-7 is not expressed in LAN human neuroblastoma cells as determined byreverse transcription-polymerase chain reaction (RT-PCR). mda-7expression is not induced by treatment with RA for 5 days, but it isinduced after 5 days growth in the medium containing phenylacetate orthe combination of phenylacetate and RA.

mda-7 is induced in human neuroblastoma cells as a function of growtharrest and induction of differentiation. Expression of mda-7 maycontribute to growth arrest and terminal differentiation in humanneuroblastoma cells.

Expression in Human Promyelocytic Leukemia Cells (HL-60) and HistiocyticLymphoma (U-937) Cell Lines

mda-7 expression is not detected in HL-60 and U-937 cells using RT-PCR.mda-7 expression is induced in HL-60 and U-937 cells following treatmentwith the growth suppressing and differentiation inducing agent TPA.mda-7 expression persists in terminally differentiated HL-60 cells aftertreatment with TPA for 2 days and RA for 4 days.

mda-7 is induced during differentiation along both granulocytic andagranulocytic (monocytic/macrophage) lineages in human promyelocyticleukemia and histiocytic lymphoma cells. Expression of mda-7 maycontribute to the growth arrest and terminal differentiation inhematopoeitic cells.

Expression in Senescent Human Cells

mda-7 expression is not detected using RT-PCR in IMR90 human cellsdisplaying proliferative potential (i.e., non-senescent cells). mda-7expression is detected in IMR90 cells grown for extended times inculture (OLD) and approaching senescence.

mda-7 gene expression inversely correlates with proliferative potentialin human cells. mda-7 expression is activated during cellularsenescence. This gene may contribute to proliferative capacity in cellsand may function as a genetic marker and/or regulatory switch ofcellular senescence.

Expression in Normal Cerebellum, a Central Nervous System Tumor(Glioblastoma Multiforme) (GBM) and Normal Skin Fibroblast Cell Lines

mda-7 is not expressed de novo in normal cerebellum, GBM or normal skinfibroblasts. Expression of mda-7 is induced in normal cerebellum, GBMand normal skin fibroblasts following a 24 hr treatment with IFN-β+MEZ.

mda-7 is not expressed de novo, but it is susceptible to induction byIFN-β+MEZ in human cerebellum, GBM and normal human skin fibroblasts.

Expression in Colorectal (SW613), Endometrial Adenocarcinoma (HTB113)and Prostate Carcinoma (LNCaP)

mda-7 is expressed de novo in colorectal carcinoma (SW613), endometrialadenocarcinoma (HTB113) or prostate carcinoma (LNCaP). mda-7 is notinduced in colorectal carcinoma (SW613), endometrial adenocarcinoma(HTB113) or prostate carcinoma (LNCaP) cells following a 24 h treatmentwith IFN-β+MEZ.

This gene is neither expressed de novo nor inducible by IFN-β+MEZ humancarcinomas.

Effect of Various Treatment Protocols on Expression in HO-1 Cells

Treatment with IFN-β (2000 units/ml; 24 h), MEZ (10 μg/ml; 24 h),IFN-β+MEZ (2000 units/ml+10 ng/ml; 24 h and 96 h), IFN-α+MEZ (2000units/ml+10 ng/ml; 24 h), adriamycin (0.1 ng/ml; 24 h), vincristine (0.1μg/ml; 24 h), and UV (10 joules/mm² and assayed 24 h later) results inincreased mda-7 expression in HO-1 cells. mda-7 is also induced after 96h treatment with MPA (2 μM), IFN-β+IFN-γ (1000 units/ml+1000 units/ml),MPA+MEZ (3 μM+10 ng/ml) and RA+MEZ (2.5 μM+10 ng/ml).

Highest level of expression observed in HO-1 cells treated withIFN-β+MEZ for 24 or 96 h.

No induction in mda-7 expression is observed in HO-1 cells treated withIFN-α (2000 units/ml; 24 h), IFN-γ (2000 units/ml; 96 h), phenylbutyrate (4 mM PB for 24 h, 4d or 7d), cis-platinum (0.1 μg/ml; 24 h),gamma irradiation (treated with 3 gray and analyzed after 24 h),actinomycin D (5 μg/ml for 2 h, assayed 24 h later), TNF-α (100units/ml; 24 h) or VP-16 (5 μg/ml; 24 h).

General Conclusions

mda-7 is a growth, differentiation-regulated and senescence-associatednovel gene which displays the following properties: 1) it is inducibleduring terminal differentiation (treatment with IFN-β+MEZ for 96 h) andfollowing treatment for 96 h with many growth modulating anddifferentiation inducing agents; 2) treatment for 24 h with IFN-β+MEZresults in increased expression in all human melanomas tested, but notin an SV40-immortalized human melanocyte; 3) it is not expressed ingrowing human neuroblastoma cells but it is inducible following growthsuppression and the induction of terminal differentiation 4) it is notexpressed in human promyelocytic leukemia (HL-60) and human histiocyticlymphoma (U-937) cells but it is induced following the induction ofgrowth arrest and terminal differentiation; 5) it is not expressed inactively growing human cells but it is induced during cellularsenescence; 6) it is not expressed de novo but it is highly inducible byIFN-β+MEZ within 24 h in normal cerebellum, GBM and normal skinfibroblast cells; 7) it is not expressed or inducible in colorectal,endometrial or prostate carcinomas; and 8) increased expression isinduced in HO-1 cells treated with adriamycin, vincristine and UVirradiation.

mda-7 is a novel growth, terminal differentiation- andsenescence-regulated gene displaying increased expression in allmelanomas (but not in melanocytes), and in normal skin fibroblasts andin both normal cerebellum and GBM cells treated with IFN-β+MEZ. mda-7 isnot expressed de novo, but it is induced during growth arrest anddifferentiation in human neuroblastoma, leukemia and histiocyticlymphoma cells. mda-7 is not expressed in growing nonsenescent cells butit is expressed in senescent cells. In contrast, mda-7 is not expressedor induced in a series of carcinomas. mda-7 may be useful; 1) as amarker for specific tissue lineage's (i.e., melanomas fromkeratinocytes) (diagnostic applications); 2) in distinguishingfibroblasts (inducible with IFN-β+MEZ) from carcinomas (non-induciblewith IFN-β+MEZ) (diagnostic applications); 3) in identifying cells thathave lost proliferative capacity and become senescent; 4) in monitoringinduction of differentiation in cancer cells resulting during thedifferentiation therapy of cancer; 5) for the identification of agentscapable of inducing growth suppression and various components of thedifferentiation process (including terminal differentiation) in humanmelanomas, neuroblastomas, leukemias and lymphomas (drug screeningprograms to identify new differentiation-inducing and chemotherapeuticagents); and 6) distinguishing melanocytes, and perhaps nevi, from earlyand late stage melanoma cells (diagnostic applications). This gene (usedin a sense orientation in an appropriate expression vector) may alsoprove useful in inhibiting growth and inducing terminal differentiationin human melanomas (therapeutic applications). When used in an antisenseorientation and inserted into bone marrow cells this gene might preventdamage resulting from chemotherapy and radiation (therapeuticapplications).

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 31                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 158 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - TGGACTTGTG TTCTGACTAG AACTCAACAT GTTACTAGGC ACATGTGTCA TG -            #TCTCAGGT     60                                                                 - - CAGTGCTGTG ACAGAATTGA TACGAGAGAA ATGTCGCTTA TGCTATCACT GA -            #TCTACACA    120                                                                 - - TGTCTGATAG ATAGTCAGAT ACAGATGATG AGGAATCT      - #                      - #    158                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 270 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - GAATTCAGTG AACTCTTTTC TCATTCTCTT TGTTTTGTGG CACTTCACAA TG -             #TAGAGGAA     60                                                                 - - AAAACCAAAT GACCGCACTG TGATGTGAAT GGCACCGAAG TCAGATGAGT AT -            #CCTGTAGG    120                                                                 - - TCACCTGCAG CCTGGCTTGC CACTTGTCTT AACTCTGAAT ATTTCATTTC AA -            #AGGTGCTA    180                                                                 - - AAATCTGAAA TCTGCTAGTG TGAACTTGCT CTACTCTCTG AATGATTCAA TC -            #CTATTCAT    240                                                                 - - ACTATCTTGT AGATATATCA ACTAAAAAAA         - #                  - #              270                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 290 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - TCCTCCTCTG CACCATGGCT CTCTGCAACC AGTTCTCTGC ATCACTTGCT GC -             #TGACACGC     60                                                                 - - CGACCGCCTG CTGCTTCAGC TACACCTCCC GGCAGATTCC ACAGAATTTC AT -            #AGCTGACT    120                                                                 - - ACTTTGAGAC GAGCAGCCAG TGCTCCAAGC CCGGTGTCAT CTTCCTAACC AA -            #GCGAACCG    180                                                                 - - GGCAGGTCTG TGCTGACCCC AGTGAGGAGT GGGTCCAGAA ATATGTCAGC GA -            #CCTGGAGC    240                                                                 - - TGAGTAGTGA GGAGTGGGTC CAGAAATATG TCAGCGACCT GGAGCTGAGT  - #                 290                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 144 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - TTCTTCTTTG TAAAAGTTTT TAATACACTG CTGAAAGATA AATTCATTCC AA -             #AGAGAATA     60                                                                 - - ATTATATAGC AAGATATTAT CGGCACAGTG GTTTCTTAGA GGTAAATAGC GC -            #CTCACGTG    120                                                                 - - TGTTAGATGC TGAATCTGAC CAAA          - #                  - #                   144                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 193 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - CTGCAAAAGA AGTGTGCCGA CTATAAATAA ATGGTGAAAT CATCTGCAAA TG -             #TGGCCAGG     60                                                                 - - CTTGGGGAAC AATGATGGTG CACAAAGGCT TAGATTTGCC TTGTCTCAAA AT -            #AAGGAATT    120                                                                 - - TTGTAGTGGT TTCAAAATAT CACAAGAACG TACAAGTGGT AGATACTATC AC -            #ATTCACTG    180                                                                 - - ACTATCAGAG TCG              - #                  - #                      - #     193                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 301 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - ACAAACCAGT GATTCCCCTT CCTCAGATAC TGGGACTAAC AGCTTCACCT GG -             #TGTTGGAG     60                                                                 - - GGGCCACGAA GCAAGCCAAA GCTGAAGAAC ACATTTTAAA ACTATGTGCC TA -            #TCTTGATG    120                                                                 - - CATTTACTAT TAAAACTGTT AAAGAAAACC TTGATCAACT GAAAAACCAA AT -            #ACAGGAGC    180                                                                 - - ATGCAAGAAG TTTGCCATTG CAGATGCAAC CAGAGAAGAT CCATTTAAAG AG -            #AAACTTCT    240                                                                 - - AGAAATAATG ACAAGGATTC AAACTTATTG TCAAATGAGT CCAATGTCAG AT -            #TTTGGACT    300                                                                 - - C                  - #                  - #                  - #                  301                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 218 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - ATGCCACGTG GGCTCATATG GGGCTGGGAG TAGTTGTCTT TCCTGGCACT AA -             #CGTTGAGC     60                                                                 - - CCCTGGAGGC ACTGAAGTGC TTAGTGTACT TGGAGTATTG GGGTCTGACC CA -            #AACACCTT    120                                                                 - - CCAGCTCCTG TAACATACTG GCCTGGACTG TTTTCTCTCG CGCCTCCCCA TG -            #TGCTCCTG    180                                                                 - - GTTCCCGTTT CCTCCACCTA GACTGTAAAC CTCTCGCA      - #                      - #    218                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 279 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - CCTGCAGTCC TGGAAGCGCG AGGGCCTCAA ACGCGCTCTA CATCTTCTGC CT -             #TAGTCTCA     60                                                                 - - GTTTGCGTGT CTTAATTATT ATTTGTGTTT TAATTTAAAC ACCTCCTCAT GT -            #ACATACCC    120                                                                 - - TGGCCGCCCC CTGCCCCCCA GCCTCTCGGA TTAGAATTAT TTAAACAAAA AC -            #TAGGCGGT    180                                                                 - - TGAATGAGAG GTTCCTATGA GTACTGGGCA TTTTTATTTT ATGAAATACT AT -            #TTAAAGCC    240                                                                 - - TCCTCATCCC ATGTTCTCCT TTTCCTCTCT CCCGGAGTT      - #                      - #   279                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 193 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - CAGAATATTG TGCCCCATGC TTCTTTACCC CTCACAATCC TTGCCACAGT GT -             #GGGCAGTG     60                                                                 - - GATGGGTGCT TAGTAAGTAC TTAATAAACT GTGGTGCTTT TTTTGGCCTG TC -            #TTTGGATT    120                                                                 - - GTTAAAAAAC AGAGAGGGAT GCTTGGATGT AAACTGAACT TCAGAGCATG AA -            #ATCACACT    180                                                                 - - GTCTCTGATA TCT              - #                  - #                      - #     193                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 182 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - TTAAAGTTTG CCCTTGTGCT AAAGTGCCAG TGTATGTATG TTATACTTGA TT -             #TGGTTGTA     60                                                                 - - AACTATATTT CAAAGTAAAC CCTAGTGTAA TAAGTTTTAT AACTAAAAAG GT -            #TTAAGCTG    120                                                                 - - CTAAAACTAT TTTTAAGAGA TGTGAAATCG AGTATGGGAC TATCTTTTTT TC -            #CTCCTCTA    180                                                                 - - AA                  - #                  - #                  - #                 182                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 216 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - AAAACTTTCA AGAGATTTAC TGACTTTCCT AGAATAGTTT CTCTACTGGA AA -             #CCTGATGC     60                                                                 - - TTTTATAAGC CATTGTGATT AGGATGACTG TTACAGGCTT AGCTTTGTGT GA -            #AAACCAGT    120                                                                 - - CACCTTTCTC CTAGGTAATG AGTAGTGCTG TTCATATTAC TTTAGTTCTA TA -            #GCATACTC    180                                                                 - - GATCTTTAAC ATGCTATCAT AGTACATTAG ATGATG      - #                       - #      216                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 257 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - CGCACGTCAC CCACCTTCCG GCGGCCGAAG ACACTGCGAC TCCGGAGACA GC -            #CCAAATAT     60                                                                 - - CCTCGGAAGA GCGCTCCCAG GAGAAACAAG CTTGACCACT ATGCTATCAT CA -            #AGTTTCCG    120                                                                 - - CTGACCACTG AGTCTGCCAT GAAGAAGATA GAAGACAACA ACACACTTGT GT -            #TCATTGTG    180                                                                 - - GATGTTAAAG CCAACAAGCA CCAGATTAAC AGCTGTGAGA GCTGTATGAC AT -            #TGATGTGC    240                                                                 - - AGTACACCTG ATCGTCT             - #                  - #                      - #  257                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 241 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - TAAAAAAATT CATTCTCTGT GGTATCCAAG AATCAGTGAA GATGCCAGTG AA -             #ACTTCAAG     60                                                                 - - CAAATCTACT TCAACACTTC ATGTATTGTG TGGGTCTGTT GTAGGGTTGC CA -            #GATGCAAT    120                                                                 - - ACAAGATTCC TGGTTAAATT TGAATTTCAG TAAACAATGA ATAGTTTTTC AT -            #TGTACATG    180                                                                 - - AAATATCAGA ACATACTTAT ATGTAAGTAT ATTATTGATG ACAAACACAA TA -            #TTTAATAT    240                                                                 - - A                  - #                  - #                  - #                  241                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 177 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - GGGGGTGAAA CTTTCCAGTT TACTGAACTC CAGACCATGC ATGTAGTCCA CT -             #CCAGAAAT     60                                                                 - - CATGCTCGCT TCCTTGGCAC ACAGTGTTCT CCTGCCAAAT GACCCTAGAC CC -            #TCTGTCCT    120                                                                 - - GCAGAGTCAG GGTGGCTTTT ACCCTGACTG TGTCGATGCA GAGTCTGCTC GA - #CAGAT           177                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 143 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - TACGATCAGA CTGTTACATT TAGCAATCAA CAGCATGGGG CGAAAAAAAA AA -             #ATCTACTT     60                                                                 - - AAAACCCTTT GTTGGAATGC TTTACACTTT CCACAGAACA GAAACTAAAA TA -            #ACTGTTTA    120                                                                 - - CATTAGTCAC AATACAGTCT CGA           - #                  - #                   143                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 base p - #airs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - ATTA                 - #                  - #                  - #                  4                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 base p - #airs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - AATAAA                 - #                  - #                  -      #            6                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - CTCCAAGTAC ACTAAGCACT            - #                  - #                      - # 20                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - TAGTTCTACC TCAGGCAGCT            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - CATGGCCTCC AAGGAGTAAG A           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - CGTCTTCACC ACCATGGAGA A           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2147 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 95..586                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - AGCTGAGGTG TGAGCAGCTG CCGAAGTCAG TTCCTTGTGG AGCCGGAGCT GG -             #GCGCGGAT     60                                                                 - - TCGCCGAGGC ACCGAGGCAC TCAGAGGAGG CGCC ATG TCA GAA C - #CG GCT GGG            112                                                                                        - #                  - #  Met Ser Glu Pro Ala Gly                             - #                  - #    1              - # 5             - - GAT GTC CGT CAG AAC CCA TGC GGC AGC AAG GC - #C TGC CGC CGC CTC TTC          160                                                                       Asp Val Arg Gln Asn Pro Cys Gly Ser Lys Al - #a Cys Arg Arg Leu Phe                        10     - #             15     - #             20                  - - GGC CCA GTG GAC AGC GAG CAG CTG AGC CGC GA - #C TGT GAT GCG CTA ATG          208                                                                       Gly Pro Val Asp Ser Glu Gln Leu Ser Arg As - #p Cys Asp Ala Leu Met                    25         - #         30         - #         35                      - - GCG GGC TGC ATC CAG GAG GCC CGT GAG CGA TG - #G AAC TTC GAC TTT GTC          256                                                                       Ala Gly Cys Ile Gln Glu Ala Arg Glu Arg Tr - #p Asn Phe Asp Phe Val                40             - #     45             - #     50                          - - ACC GAG ACA CCA CTG GAG GGT GAC TTC GCC TG - #G GAG CGT GTG CGG GGC          304                                                                       Thr Glu Thr Pro Leu Glu Gly Asp Phe Ala Tr - #p Glu Arg Val Arg Gly            55                 - # 60                 - # 65                 - # 70       - - CTT GGC CTG CCC AAG CTC TAC CTT CCC ACG GG - #G CCC CGG CGA GGC CGG          352                                                                       Leu Gly Leu Pro Lys Leu Tyr Leu Pro Thr Gl - #y Pro Arg Arg Gly Arg                            75 - #                 80 - #                 85              - - GAT GAG TTG GGA GGA GGC AGG CGG CCT GGC AC - #C TCA CCT GCT CTG CTG          400                                                                       Asp Glu Leu Gly Gly Gly Arg Arg Pro Gly Th - #r Ser Pro Ala Leu Leu                        90     - #             95     - #            100                  - - CAG GGG ACA GCA GAG GAA GAC CAT GTG GAC CT - #G TCA CTG TCT TGT ACC          448                                                                       Gln Gly Thr Ala Glu Glu Asp His Val Asp Le - #u Ser Leu Ser Cys Thr                   105          - #       110          - #       115                      - - CTT GTG CCT CGC TCA GGG GAG CAG GCT GAA GG - #G TCC CCA GGT GGA CCT          496                                                                       Leu Val Pro Arg Ser Gly Glu Gln Ala Glu Gl - #y Ser Pro Gly Gly Pro               120              - #   125              - #   130                          - - GGA GAC TCT CAG GGT CGA AAA CGG CGG CAG AC - #C AGC ATG ACA GAT TTC          544                                                                       Gly Asp Ser Gln Gly Arg Lys Arg Arg Gln Th - #r Ser Met Thr Asp Phe           135                 1 - #40                 1 - #45                 1 -      #50                                                                              - - TAC CAC TCC AAA CGC CGG CTG ATC TTC TCC AA - #G AGG AAG CCC                 - # 586                                                                   Tyr His Ser Lys Arg Arg Leu Ile Phe Ser Ly - #s Arg Lys Pro                                   155  - #               160                                     - - TAATCCGCCC ACAGGAAGCC TGCAGTCCTG GAAGCGCGAG GGCCTCAAAG GC -             #CCGCTCTA    646                                                                 - - CATCTTCTGC CTTAGTCTCA GTTTGTGTGT CTTAATTATT ATTTGTGTTT TA -            #ATTTAAAC    706                                                                 - - ACCTCCTCAT GTACATACCC TGGCCGCCCC CTGCCCCCCA GCCTCTGGCA TT -            #AGAATTAT    766                                                                 - - TTAAACAAAA ACTGGGCGGT TGAATGAGAG GTTCCTAAGA GTGCTGGGCA TT -            #TTTATTTT    826                                                                 - - ATGAAATACT ATTTAAAGCC TCCTCATCCC GTGTTCTCCT TTTCCTCTCT CC -            #CGGAGGTT    886                                                                 - - GGGTGGGCCG GCTTCATGCC AGCTACTTCC TCCTCCCCAC TTGTCCGCTG GG -            #TGGTACCC    946                                                                 - - TCTGGAGGGG TGTGGCTCCT TCCCATCGCT GTCACAGGCG GTTATGAAAT TC -            #ACCCCCTT   1006                                                                 - - TCCTGGACAC TCAGACCTGA ATTCTTTTTC ATTTGAGAAG TAAACAGATG GC -            #ACTTTGAA   1066                                                                 - - GGGGCCTCAC CGAGTGGGGG CATCATCAAA AACTTTGGAG TCCCCTCACC TC -            #CTCTAAGG   1126                                                                 - - TTGGGCAGGG TGACCCTGAA GTGAGCACAG CCTAGGGCTG AGCTGGGGAC CT -            #GGTACCCT   1186                                                                 - - CCTGGCTCTT GATACCCCCC TCTGTCTTGT GAAGGCAGGG GGAAGGTGGG GT -            #CCTGGAGC   1246                                                                 - - AGACCACCCC GCCTGCCCTC ATGGCCCCTC TGACCTGCAC TGGGGAGCCC GT -            #CTCAGTGT   1306                                                                 - - TGAGCCTTTT CCCTCTTTGG CTCCCCTGTA CCTTTTGAGG AGCCCCAGCT AC -            #CCTTCTTC   1366                                                                 - - TCCAGCTGGG CTCTGCAATT CCCCTCTGCT GCTGTCCCTC CCCCTTGTCC TT -            #TCCCTTCA   1426                                                                 - - GTACCCTCTC AGCTCCAGGT GGCTCTGAGG TGCCTGTCCC ACCCCCACCC CC -            #AGCTCAAT   1486                                                                 - - GGACTGGAAG GGGAAGGGAC ACACAAGAAG AAGGGCACCC TAGTTCTACC TC -            #AGGCAGCT   1546                                                                 - - CAAGCAGCGA CCGCCCCCTC CTCTAGCTGT GGGGGTGAGG GTCCCATGTG GT -            #GGCACAGG   1606                                                                 - - CCCCCTTGAG TGGGGTTATC TCTGTGTTAG GGGTATATGA TGGGGGAGTA GA -            #TCTTTCTA   1666                                                                 - - GGAGGGAGAC ACTGGCCCCT CAAATCGTCC AGCGACCTTC CTCATCCACC CC -            #ATCCCTCC   1726                                                                 - - CCAGTTCATT GCACTTTGAT TAGCAGCGGA ACAAGGAGTC AGACATTTTA AG -            #ATGGTGGC   1786                                                                 - - AGTAGAGGCT ATGGACAGGG CATGCCACGT GGGCTCATAT GGGGCTGGGA GT -            #AGTTGTCT   1846                                                                 - - TTCCTGGCAC TAACGTTGAG CCCCTGGAGG CACTGAAGTG CTTAGTGTAC TT -            #GGAGTATT   1906                                                                 - - GGGGTCTGAC CCCAAACACC TTCCAGCTCC TGTAACATAC TGGCCTGGAC TG -            #TTTTCTCT   1966                                                                 - - CGGCTCCCCA TGTGTCCTGG TTCCCGTTTC TCCACCTAGA CTGTAAACCT CT -            #CGAGGGCA   2026                                                                 - - GGGACCACAC CCTGTACTGT TCTGTGTCTT TCACAGCTCC TCCCACAATG CT -            #GAATAAAC   2086                                                                 - - AGCAGGTGCT CAATAAATGA TTCTTAGTGA CTTTAAAAAA AAAAAAAAAA AA -            #AAAAAAAA   2146                                                                 - - A                  - #                  - #                  - #                 2147                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 164 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - Met Ser Glu Pro Ala Gly Asp Val Arg Gln As - #n Pro Cys Gly Ser Lys        1               5 - #                 10 - #                 15              - - Ala Cys Arg Arg Leu Phe Gly Pro Val Asp Se - #r Glu Gln Leu Ser Arg                   20     - #             25     - #             30                  - - Asp Cys Asp Ala Leu Met Ala Gly Cys Ile Gl - #n Glu Ala Arg Glu Arg               35         - #         40         - #         45                      - - Trp Asn Phe Asp Phe Val Thr Glu Thr Pro Le - #u Glu Gly Asp Phe Ala           50             - #     55             - #     60                          - - Trp Glu Arg Val Arg Gly Leu Gly Leu Pro Ly - #s Leu Tyr Leu Pro Thr       65                 - # 70                 - # 75                 - # 80       - - Gly Pro Arg Arg Gly Arg Asp Glu Leu Gly Gl - #y Gly Arg Arg Pro Gly                       85 - #                 90 - #                 95              - - Thr Ser Pro Ala Leu Leu Gln Gly Thr Ala Gl - #u Glu Asp His Val Asp                  100      - #           105      - #           110                  - - Leu Ser Leu Ser Cys Thr Leu Val Pro Arg Se - #r Gly Glu Gln Ala Glu              115          - #       120          - #       125                      - - Gly His Val Asp Leu Ser Leu Ser Cys Thr Le - #u Val Pro Arg Ser Gly          130              - #   135              - #   140                          - - Glu Gln Ala Glu Gly Ser Pro Gly Gly Pro Gl - #y Asp Ser Gln Gly Arg      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Arg Arg Gln                                                           - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 495 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..491                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - ATG TCA GAA CCG GCT GGG GAT GTC CGT CAG AA - #C CCA TGC GGC AGC        AAG       48                                                                    Met Ser Glu Pro Ala Gly Asp Val Arg Gln As - #n Pro Cys Gly Ser Lys          165                 1 - #70                 1 - #75                 1 -      #80                                                                              - - GCC TGC CGC CGC CTC TTC GGC CCA GTG GAC AG - #C GAG CAG CTG AGC        CGC       96                                                                    Ala Cys Arg Arg Leu Phe Gly Pro Val Asp Se - #r Glu Gln Leu Ser Arg                          185  - #               190  - #               195              - - GAC TGT GAT GCG CTA ATG GCG GGC TGC ATC CA - #G GAG GCC CGT GAG CGA          144                                                                       Asp Cys Asp Ala Leu Met Ala Gly Cys Ile Gl - #n Glu Ala Arg Glu Arg                       200      - #           205      - #           210                  - - TGG AAC TTC GAC TTT GTC ACC GAG ACA CCA CT - #G GAG GGT GAC TTC GCC          192                                                                       Trp Asn Phe Asp Phe Val Thr Glu Thr Pro Le - #u Glu Gly Asp Phe Ala                   215          - #       220          - #       225                      - - TGG GAG CGT GTG CGG GGC CTT GGC CTG CCC AA - #G CTC TAC CTT CCC ACG          240                                                                       Trp Glu Arg Val Arg Gly Leu Gly Leu Pro Ly - #s Leu Tyr Leu Pro Thr               230              - #   235              - #   240                          - - GGG CCC CGG CGA GGC CGG GAT GAG TTG GGA GG - #A GGC AGG CGG CCT GGC          288                                                                       Gly Pro Arg Arg Gly Arg Asp Glu Leu Gly Gl - #y Gly Arg Arg Pro Gly           245                 2 - #50                 2 - #55                 2 -      #60                                                                              - - ACC TCA CCT GCT CTG CTG CAG GGG ACA GCA GA - #G GAA GAC CAT GTG        GAC      336                                                                    Thr Ser Pro Ala Leu Leu Gln Gly Thr Ala Gl - #u Glu Asp His Val Asp                          265  - #               270  - #               275              - - CTG TCA CTG TCT TGT ACC CTT GTG CCT CGC TC - #A GGG GAG CAG GCT GAA          384                                                                       Leu Ser Leu Ser Cys Thr Leu Val Pro Arg Se - #r Gly Glu Gln Ala Glu                       280      - #           285      - #           290                  - - GGG TCC CCA GGT GGA CCT GGA GAC TCT CAG GG - #T CGA AAA CGG CGG CAG          432                                                                       Gly Ser Pro Gly Gly Pro Gly Asp Ser Gln Gl - #y Arg Lys Arg Arg Gln                   295          - #       300          - #       305                      - - ACC AGC ATG ACA GAT TTC TAC CAC TCC AAA CG - #C CGG CTG ATC TTC TCC          480                                                                       Thr Ser Met Thr Asp Phe Tyr His Ser Lys Ar - #g Arg Leu Ile Phe Ser               310              - #   315              - #   320                          - - AAG AGG AAG  CCC TAA          - #                  - #                      - #   495                                                                  Lys Arg Lys                                                                   325                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 163 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - Met Ser Glu Pro Ala Gly Asp Val Arg Gln As - #n Pro Cys Gly Ser Lys        1               5 - #                 10 - #                 15              - - Ala Cys Arg Arg Leu Phe Gly Pro Val Asp Se - #r Glu Gln Leu Ser Arg                   20     - #             25     - #             30                  - - Asp Cys Asp Ala Leu Met Ala Gly Cys Ile Gl - #n Glu Ala Arg Glu Arg               35         - #         40         - #         45                      - - Trp Asn Phe Asp Phe Val Thr Glu Thr Pro Le - #u Glu Gly Asp Phe Ala           50             - #     55             - #     60                          - - Trp Glu Arg Val Arg Gly Leu Gly Leu Pro Ly - #s Leu Tyr Leu Pro Thr       65                 - # 70                 - # 75                 - # 80       - - Gly Pro Arg Arg Gly Arg Asp Glu Leu Gly Gl - #y Gly Arg Arg Pro Gly                       85 - #                 90 - #                 95              - - Thr Ser Pro Ala Leu Leu Gln Gly Thr Ala Gl - #u Glu Asp His Val Asp                  100      - #           105      - #           110                  - - Leu Ser Leu Ser Cys Thr Leu Val Pro Arg Se - #r Gly Glu Gln Ala Glu              115          - #       120          - #       125                      - - Gly Ser Pro Gly Gly Pro Gly Asp Ser Gln Gl - #y Arg Lys Arg Arg Gln          130              - #   135              - #   140                          - - Thr Ser Met Thr Asp Phe Tyr His Ser Lys Ar - #g Arg Leu Ile Phe Ser      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Arg Lys                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1720 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 374..891                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - CTTGCCTGCA AACCTTTACT TCTGAAATGA CTTCCACGGC TGGGACGGGA AC -            #CTTCCACC     60                                                                 - - CACAGCTATG CCTCTGATTG GTGAATGGTG AAGGTGCCTG TCTAACTTTT CT -            #GTAAAAAG    120                                                                 - - AACCAGCTGC CTCCAGGCAG CCAGCCCTCA AGCATCACTT ACAGGACCAG AG -            #GGACAAGA    180                                                                 - - CATGACTGTG ATGAGGAGCT GCTTTCGCCA ATTTAACACC AAGAAGAATT GA -            #GGCTGCTT    240                                                                 - - GGGAGGAAGG CCAGGAGGAA CACGAGACTG AGAGATGAAT TTTCAACAGA GG -            #CTGCAAAG    300                                                                 - - CCTGTGGACT TTAGCCAGAC CCTTCTGCCC TCCTTTGCTG GCGACAGCCT CT -            #CAAATGCA    360                                                                 - - GATGGTTGTG CTC CCT TGC CTG GGT TTT ACC CTG CT - #T CTC TGG AGC CAG           409                                                                                     Pro Cys - #Leu Gly Phe Thr Leu Leu Leu Trp Ser Gln                               - # 165                - # 170                - # 175        - - GTA TCA GGG GCC CAG GGC CAA GAA TTC CAC TT - #T GGG CCC TGC CAA GTG          457                                                                       Val Ser Gly Ala Gln Gly Gln Glu Phe His Ph - #e Gly Pro Cys Gln Val                           180  - #               185  - #               190              - - AAG GGG GTT GTT CCC CAG AAA CTG TGG GAA GC - #C TTC TGG GCT GTG AAA          505                                                                       Lys Gly Val Val Pro Gln Lys Leu Trp Glu Al - #a Phe Trp Ala Val Lys                       195      - #           200      - #           205                  - - GAC ACT ATG CAA GCT CAG GAT AAC ATC ACG AG - #T GCC CGG CTG CTG CAG          553                                                                       Asp Thr Met Gln Ala Gln Asp Asn Ile Thr Se - #r Ala Arg Leu Leu Gln                   210          - #       215          - #       220                      - - CAG GAG GTT CTG CAG AAC GTC TCG GAT GCT GA - #G AGC TGT TAC CTT GTC          601                                                                       Gln Glu Val Leu Gln Asn Val Ser Asp Ala Gl - #u Ser Cys Tyr Leu Val               225              - #   230              - #   235                          - - CAC ACC CTG CTG GAG TTC TAC TTG AAA ACT GT - #T TTC AAA AAC TAC CAC          649                                                                       His Thr Leu Leu Glu Phe Tyr Leu Lys Thr Va - #l Phe Lys Asn Tyr His           240                 2 - #45                 2 - #50                 2 -      #55                                                                              - - AAT AGA ACA GTT GAA GTC AGG ACT CTG AAG TC - #A TTC TCT ACT CTG        GCC      697                                                                    Asn Arg Thr Val Glu Val Arg Thr Leu Lys Se - #r Phe Ser Thr Leu Ala                          260  - #               265  - #               270              - - AAC AAC TTT GTT CTC ATC GTG TCA CAA CTG CA - #A CCC AGT CAA GAA AAT          745                                                                       Asn Asn Phe Val Leu Ile Val Ser Gln Leu Gl - #n Pro Ser Gln Glu Asn                       275      - #           280      - #           285                  - - GAG ATG TTT TCC ATC AGA GAC AGT GCA CAC AG - #G CGG TTT CTG CTA TTC          793                                                                       Glu Met Phe Ser Ile Arg Asp Ser Ala His Ar - #g Arg Phe Leu Leu Phe                   290          - #       295          - #       300                      - - CGG AGA GCA TTC AAA CAG TTG GAC GTA GAA GC - #A GCT CTG ACC AAA GCC          841                                                                       Arg Arg Ala Phe Lys Gln Leu Asp Val Glu Al - #a Ala Leu Thr Lys Ala               305              - #   310              - #   315                          - - CTT GGG GAA GTG GAC ATT CTT CTG ACC TGG AT - #G CAG AAA TTC TAC AAG     CT   891                                                                       Leu Gly Glu Val Asp Ile Leu Leu Thr Trp Me - #t Gln Lys Phe Tyr Lys           320                 3 - #25                 3 - #30                 3 -      #35                                                                              - - CTCTGAATGT CTAGACCAGG ACCTCCCTCC CCCTGGCACT GGTTTGTTCC CT -            #GTGTCATT    951                                                                 - - TCAAACAGTC TCCCTTCCTA TGCTGTTCAC TGGACACTTC ACGCCCTTGG CC -            #ATGGGTCC   1011                                                                 - - CATTCTTGGC CCAGGATTAT TGTCAAAGAA GTCATTCTTT AAGCAGCGCC AG -            #TGACAGTC   1071                                                                 - - AGGGAAGGTG CCTCTGGATG CTGTGAAGAG TCTACAGAGA AGATTCTTGT AT -            #TTATTACA   1131                                                                 - - ACTCTATTTA ATTAATGTCA GTATTTCAAC TGAAGTTCTA TTTATTTGTG AG -            #ACTGTAAG   1191                                                                 - - TTACATGAAG GCAGCAGAAT ATTGTGCCCC ATGCTTCTTT ACCCCTCACA AT -            #CCTTGCCA   1251                                                                 - - CAGTGTGGGG CAGTGGATGG GTGCTTAGTA AGTACTTAAT AAACTGTGGT GC -            #TTTTTTTG   1311                                                                 - - GCCTGTCTTT GGATTGTTAA AAAACAGAGA GGGATGCTTG GATGTAAAAC TG -            #AACTTCAG   1371                                                                 - - AGCATGAAAA TCACACTGTC TGCTGATATC TGCAGGGACA GAGCATTGGG GT -            #GGGGGTAA   1431                                                                 - - GGTGCATCTG TTTGAAAAGT AAACGATAAA ATGTGGATTA AAGTGCCCAG CA -            #CAAAGCAG   1491                                                                 - - ATCCTCAATA AACATTTCAT TTCCCACCCA CACTCGCCAG CTCACCCCAT CA -            #TCCCTTTC   1551                                                                 - - CCTTGGTGCC CTCCTTTTTT TTTTATCCTA GTCATTCTTC CCTAATCTTC CA -            #CTTGAGTG   1611                                                                 - - TCAAGCTGAC CTTGCTGATG GTGACATTGC ACCTGGATGT ACTATCCAAT CT -            #GTGATGAC   1671                                                                 - - ATTCCCTGCT AATAAAAGAC AACATAACTC AAAAAAAAAA AAAAAAAAA  - #                 1720                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 172 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - Pro Cys Leu Gly Phe Thr Leu Leu Leu Trp Se - #r Gln Val Ser Gly Ala        1               5 - #                 10 - #                 15              - - Gln Gly Gln Glu Phe His Phe Gly Pro Cys Gl - #n Val Lys Gly Val Val                   20     - #             25     - #             30                  - - Pro Gln Lys Leu Trp Glu Ala Phe Trp Ala Va - #l Lys Asp Thr Met Gln               35         - #         40         - #         45                      - - Ala Gln Asp Asn Ile Thr Ser Ala Arg Leu Le - #u Gln Gln Glu Val Leu           50             - #     55             - #     60                          - - Gln Asn Val Ser Asp Ala Glu Ser Cys Tyr Le - #u Val His Thr Leu Leu       65                 - # 70                 - # 75                 - # 80       - - Glu Phe Tyr Leu Lys Thr Val Phe Lys Asn Ty - #r His Asn Arg Thr Val                       85 - #                 90 - #                 95              - - Glu Val Arg Thr Leu Lys Ser Phe Ser Thr Le - #u Ala Asn Asn Phe Val                  100      - #           105      - #           110                  - - Leu Ile Val Ser Gln Leu Gln Pro Ser Gln Gl - #u Asn Glu Met Phe Ser              115          - #       120          - #       125                      - - Ile Arg Asp Ser Ala His Arg Arg Phe Leu Le - #u Phe Arg Arg Ala Phe          130              - #   135              - #   140                          - - Lys Gln Leu Asp Val Glu Ala Ala Leu Thr Ly - #s Ala Leu Gly Glu Val      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Asp Ile Leu Leu Thr Trp Met Gln Lys Phe Ty - #r Lys                                      165  - #               170                                     - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - CTCCAAGTAC ACTAAGCACT            - #                  - #                      - # 20                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - - TAGTTCTACC TCAGGCAGCT            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - - TCTTACTCCT TGGAGGCCAT G           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - - CGTCTTCACC ACCACCATGG AGAA          - #                  - #                    24                                                                    __________________________________________________________________________

What is claimed is:
 1. A method for identifying an anticancer compoundcomprising:a) contacting the human melanoma, leukemia, lung tumor, braintumor, liver tumor or colon tumor cells with an appropriate amount ofthe compound; and b) measuring the expression level of mda-6 gene in thecells, an increase of the expression level indicating that the compoundis an anticancer compound.
 2. A method for identifying a growthsuppression compound comprising:a) contacting human melanoma, leukemia,lung tumor, brain tumor, liver tumor or colon tumor cells with anappropriate amount of the compound; and b) measuring the expressionlevel of mda-6 gene in the cells, an increase of the expression levelindicating that the compound is a growth suppression compound.
 3. Amethod for identifying a DNA damaging compound comprising:a) contactinghuman melanoma, leukemia, lung tumor, brain tumor, liver tumor or colontumor cells with an appropriate amount of the compound; and b) measuringthe expression level of mda-6 gene in the cells, an increase of theexpression level indicating that the compound is a DNA damagingcompound.
 4. A method for monitoring response to topoisomerase inhibitorby a human melanoma, leukemia, lung tumor, brain tumor, liver tumor orcolon tumor cell comprising detecting the expression of mda-6, theexpression of mda-6 indicating that the cell responds to thetopoisomerase inhibitor.
 5. A method for identifying a compound capableof inducing terminal differentiation in human, melanoma, leukemia, lungtumor, brain tumor, liver tumor or colon tumor cells comprising:a)incubating said cells with an appropriate concentration of the compound;and b) detecting the expression of mda-6, the expression of mda-6 geneindicating that the compound is capable of inducing terminaldifferentiation in said cells.
 6. The method of claim 5, wherein thecells are neuroblastoma cells or glioblastoma multiforme cells.
 7. Amethod for indicating the effectiveness of a treatment against cancercomprising measuring the expression level of mda-6 gene in the humanmelanoma, leukemia lung tumor, brain tumor, liver tumor or colon tumorcells, the increase of the expression level indicating the effectivenessof the treatment.
 8. The method of claim 7, wherein the cancer is aneuroblastoma or a glioblastoma multiforme tumor.